Vaccines against animal parasitic nematodes

ABSTRACT

Disclosed is the invention relates to proteins derived from parasitic nematodes which confer protective immunity against infection by parasitic nematodes, to nucleotide sequences encoding these proteins, to recombinant molecules containing such sequences to host cells transformed with such recombinant molecules and methods for the production of the nucleotide sequences recombinant molecules and hosts. The invention also relates to vaccines comprising proteins of the invention together with suitable carriers or diluents and to antibodies raised against proteins of the invention.

This application is a divisional of Ser. No. 07/353,658 filed May 2, 1989 which is a 371 of PCT/AU88/00239 filed Jul. 6, 1988.

TECHNICAL FIELD

The invention relates to the identification of proteins that confer protective immunity against infection by parasitic nematodes such as Trichinella spiralis or Ancylostoma canium infections of man, Strongylus vulgaris infections of horses, Trichostrongylus colubriformis infections of sheep, Haemonchus contortus infections of sheep and goats, Ostertagia ostertagi infections of cattle, Ascaris suum or Trichinella spiralis infections of pigs, Toxascaris leonia or Uncinaria stenocephala infections of cats, Ancylostoma canium or Trichuris vulpis infections of dogs, Dirofilaria immitis infections of dogs, infections of man by larvae of Toxocara spp. and infection by Necator americanus, Ancylostoma duodenala, Ascaris lumbricoides, Trichuris trichiura, Enterobius vermicularus, Strongyloides stercorals or Wucheraria bancrofti.

The invention further provides nucleotide sequences encoding these proteins or derivatives thereof as well as recombinant molecules containing such nucleotide sequences and host cells expressing these nucleotide sequences. The invention also provides methods for the production of the nucleotide sequences, recombinant molecules and hosts of the invention.

Additionally, the invention relates to vaccines conferring protective immunity against infection by parasitic nematodes such as Trichinella spiralis or Ancylostoma canium infections of man, Strongylus vulgaris infections of horses, Trichostrongylus colubriformis infections of sheep, Haemonchus contortus infections of sheep and goats, Ostertagia ostertagi infections of cattle, Ascaris suum or Trichinella spiralis infections of pigs, Toxascaris leonia or Uncinaria stenocephala infections of cats, Ancylostoma canium or Trichuris vulpis infections of dogs, Dirofilaria immitis infections of dogs, infections of man by larvae of Toxocara spp. and infection by Necator americanus, Ancylostoma duodenala, Ascaris lumbricoides, Trichuris trichiura, Enterobius vermicularus, Strongyloides stercorals or Wucheraria bancrofti.

BACKGROUND ART

Nematodes (nema--thread; oides--resembling), which are unsegmented roundworms with elongated, fusiform, or saclike bodies covered with cuticle, are virtually ubiquitous in nature, inhabiting soil, water and plants, and are importantly involved in a wide range of animal and plant parasitic diseases.

The roundworm parasites of mammals belong to the phylum Hemathelminthes. The roundworms include the hookworm (e.g. Necator americanus and Ancylostoma duodenale), roundworm (e.g. the common roundworm Ascaris lumbricoides), whipworm (e.g. Trichuris trichiura), and the pinworm or threadworm (e.g. Enterobius vermicularus), as well as Strongyloides stercorals, Trichinella spiralis (infection in man and pigs), and the filarial worm Wuchereria bancrofti. Other important roundworm parasites include Ancylostoma canium (infections of man), Strongylus vulgaris (infections of horses), Trichostrongylus colubriformis (infections of sheep), Haemonchus contortus (infections of sheep and goats), Ostertagia ostergati (infections of cattle), Ascaris suum, Toxascaris leonia or Uncinaria stenocephala (infections of dogs), Toxocara spp. (circulatory infections of man) and Dirofilaria immitis (circulatory infections of cats and dogs).

Even when symptom-free, parasitic worm infections are harmful to the host animal for a number of reasons; e.g. they deprive the host of food, injure organs or obstruct ducts, may elaborate substances toxic to the host, and provide a port of entry for other organisms. In other cases, the host may be a species raised for food and the parasite may be transmitted upon eating to infect the ingesting animal. It is highly desirable to eliminate such parasites as soon as they have been discovered.

More commonly, such infections are not symptom-free. Helminth infections of mammals, particularly by parasitic nematodes, are a source of great economic loss, especially of livestock and pets, e.g. sheep, cattle, horses, pigs, goats, dogs, cats, and birds, especially poultry (see CSIRO/BAE Report--"Socio-economic Developments and Trends in the Agricultural Sector: Implications for Future Research". These animals must be regularly treated with antihelminthic chemicals in order to keep such infections under control, or else the disease may result in anemia, diarrhea, dehydration, loss of appetite, and even death.

The only currently available means for controlling helminth infections is with the use of antihelminthic chemicals, but these are only effective against resident worms present at the time of treatment. Therefore, treatment must be continuous since the animals are constantly exposed to infection; e.g. antihelminthic treatment with diethylcarbamazine is required every day or every other day most of the year to control Dirofilaria immitis or the dog heartworm. This is an expensive and labor intensive procedure. Due to the widespread use of anthelminthic chemicals, the worms may develop resistance and to new and more potent classes of chemicals must be developed. An alternative approach is clearly desirable.

The development of a vaccine against parasitic nematodes would overcome many of the drawbacks inherent with chemical treatment for the prevention and curing of helminthic infections. The protection would certainly last longer, only the vaccinated animal would be affected, and the problems of toxicity and persistence of residues would be minimized or avoided. Accordingly, there have been several attempts reported in the prior art to develop such vaccines using parasitic nematodes; unfortunately, they have met with limited success and factors such as material availability and vaccine stability have precluded their large scale use.

One such application described by J. K. Dineen et al., (1977) involves the use of irradiated larval vaccines. As with other such attempts, the utility of this method is restricted by the requirement to maintain viable nematodes for prolonged periods.

The failure of killed vaccine preparations to afford good antihelminthic protection has been thought to be due to a number of factors. For example, it has been considered by J. T. M. Neilson, (1975) that parasitic nematodes may have evolved mechanisms by which they can secrete products which immunosuppress or immunomodule the host's immune system, thereby both preventing the development of an effective immune response and rendering the host susceptible to other infections. It is believed by Dineen and Wagland (1982), that immunosuppressants or immunodulators may be present in the crude preparations of parasitic nematodes which are used in the killed vaccines. A second problem suggested by this review article is that parasitic nematodes may have altered their antigen profile to one which resembles that of the host so that, in a natural infection, vigorous immunological reactions are not provoked by protective parasitic antigens. Such a phenomen would also occur following vaccination with impure preparations of killed nematodes or extracts thereof.

Recent advances in biotechnology and in particular recombinant DNA technology, realistically offer the opportunity to produce commercially-viable vaccines against a range of economically-important parasites of man and domestic animals. Although it has been demonstrated (e.g. O'Donnell et al 1985) that many parasite proteins are recognised by host animals during parasitic infection, many of the immune responses will have no functional significance in terms of resistance to re-infection. The major step is to identify, from the many thousands of proteins present in the parasitic organism, the individual proteins that can induce immune responses in the host animal that protect it from re-infection. Once identified, recombinant DNA technology could be used to construct microorganisms which synthesize those proteins or portions of the proteins containing protective epitopes and use the products synthesized by the recombinant organism in vaccines to protect animals from infection with the parasites. This approach would overcome many of the problems proposed to account for the lack of efficacy of killed vaccines using crude parasite preparations discussed above. For example, the vaccines produced by recombinant DNA techniques would not contain immunosuppressants or immunomodulators which may be found in crude extracts of parasitic nematode species.

The CSIRO/BAE working paper "Socio-economic Developments and Trends in the Agricultural Sector: Implications for Future Research" cited intestinal parasites as one of the three most urgent health problems in the Australian sheep industry and indicated that the development of vaccines holds great promise for better control of these infections.

Trichostrongylus colubriformis and Haemonchus contortus are two of the most important parasites of sheep. Several attempts have been made to vaccinate animals against infection with these species of parasite (see Adams and Cobon for Review).

For example, Rothwell and co-workers (1974, 1977, 1978) have done much work to show that the whole homogenate from 4th stage larvae of T. colubriformis caused accelerated expulsion of the worms from outbred guinea pigs, a laboratory model for sheep. Sub-fractions of this whole homogenate isolated by electrochoresis in polyacrylamide gels containing sodium dodecyl sulphate (SDS-PAGE) were later also shown to cause this accelerated expulsion (O'Donnell et al 1985). It has recently been reported that a high molecular weight fraction obtained from a somatic extract of H. contortus and excretions and secretions of larvae isolated during in vitro development could be used in a vaccine and resulted in 59% reduction in the adult worm numbers compared with controls (Neilson and Van de Walle, 1987). Silverman makes similar claims in U.S. Pat. No. 894,603 and Australian patent 247 354.

However in all of these reports, crude extracts have been used and no defined antigen or individual component of the extracts have been identified as being responsible for protection. The only nematode-host system where defined-antigens have proven host-protective to date is the Trichinella spiralis-mouse system studied by Despommier and colleagues (see Silverstein and Despommier 1985). Two antigens of molecular weights 48 kD and 50-55 kD have given host-protection of 68% and 39% respectively. In addition, it has been claimed (Munn and Greenwood, 1987) that a molecule referred to as contortin isolated from H. contortus can be used in a vaccine to reduce parasitism in sheep. However this material is not characterised at the molecular level and the means by which the fraction is prepared are such that it is highly likely that the preparation is very impure and contains many components. It has not been demonstrated which component is responsible for the marginal effects observed.

DESCRIPTION OF THE INVENTION

In the present invention, a molecule termed 41 kD is described which is isolated from T. colubriformis. When prepared in a non-native form, this molecule gives 43-51% protection to guinea pigs from infection by T. colubriformis. The gene coding for the 41 kD protein has been cloned from T. colubriformis and the closely related gene from H. contortus has also been cloned. Hybridisation studies and DNA sequence analysis of the two genes show that they are closely related. A polypeptide produced by recombinant organisms containing DNA sequences from T. colubriformis is capable of giving protection to guinea pigs against parasitism by T. colubriformis. In addition, the polypeptide produced from recombinant organisms containing DNA sequences from H. contortus is capable of giving protection to sheep against H. contortus infection and is also capable of giving protection to guinea pigs against T. colubriformis infection. Thus it can be confidently predicted that protection can be obtained in other animal species against a range of parasitic nematode species by vaccination with the recombinant proteins described in this invention or by proteins produced by recombinant DNA technology using related gene products from those other parasite species. The protein and DNA sequence analysis indicates that the proteins are closely related and are expected to be present in all other related forms of parasitic nematode species. By analogy the invention is extended to cover all major animal nematode parasites.

To describe the invention in more detail, third stage larvae of T. colubriformis were extensively washed with phosphate buffered saline (PBS) and then extracted with PBS buffer containing sodium deoxycholate (PBS-DOC). The PBS-DOC extract was found to contain only a few major protein bands. These proteins have now been isolated and their ability to cause accelerated expulsion of T. colubriformis from guinea pigs examined. It has been demonstrated that one of the proteins, referred to as the 41 kD protein elicits immunity to T. colubriformis infection following intraperitoneal injection.

The 41 kD protein has been purified from third stage larvae of T. colubriformis digested with Armillaria mellea protease, the peptide fragments resolved and partial amino acid sequences of these peptides determined. Oligonucleotide sequences suitable for hybridization probes have been designed and used to identify recombinant bacterial cells containing the gene coding for the T. colubriformis 41 kD protein. The DNA sequences of this gene has been determined. Recombinant organisms have been constructed which synthesize portions of the T. colubriformis 41 kD protein and the recombinant fusion protein isolated from the bacterial cells when used to vaccinate guinea pigs has been shown to cause accelerated rejection of T. colubriformis challenge infections.

Using DNA coding for the 41 kD protein of T. colubriformis as a hybridization probe, the presence of a gene encoding a related antigen in a cDNA library constructed from mRNA extracted from young adult H. contortus has been identified. The protein produced from this gene is recognised by antiserum raised against the T. colubriformis 41 kD protein.

The H. contortus DNA sequence has been cloned and bacteria have been constructed to express this protein. The protein purified from these bacteria has been used to vaccinate sheep. These sheep were subsequently challenged with H. contortus and the animals have been shown to be significantly protected from parasitism. In addition, the protein purified from bacteria expressing the H. contortus 41 kD peptide was used to vaccinate guinea pigs, which were subsequently infected with T. colubriformis and shown to be significantly protected from parasitism. These experiments demonstrate that the approaches used to clone, and express the T. colubriformis and H. contortus antigens were also applicable to providing vaccines against other species of parasitic nematodes such as Trichinella spiralis, Ancylostoma canium, Strongylus vulgaris, Ostertagia ostertagi, Ascaris suum, Trichinella spiralis, Toxascaris leonina, Uncinaria stenocephala, Ancylostoma canium, Trichuris vulpis, Dirofilaria immitis, larvae of Toxocara spp. Necator americanus, Ancylostoma duodenale, Ascaris lumbricoides, Trichuris trichiura, Enterobius vermicularus, Strongyloides stercorals or Wuchereria bancrofti.

In a first embodiment the invention provides a protein derived from a parasitic nematode species and capable of conferring protective immunity against infection of a host by a parasitic nematode species wherein said protein has an approximate molecular weight of 41 kD, as estimated by SDS-PAGE, or all, part, an analogue, homologue or derivative or combination thereof of said protein which is capable of conferring protective immunity against infection of a host by a parasitic nematode.

Preferably said parasitic nematode is Trichinella spiralis, Ancylostoma canium, Strongylus vulgaris, Ostertagia ostertagi, Ascaris suum, Trichinella spiralis, Toxascaris leonina, Uncinaria stenocephala, Ancylostoma canium, Trichuris vulpis, Dirofilaria immitis, larvae of Toxocara spp. T. colubriformis, H. contortus, Necator americanus, Ancylostoma duodenale, Ascaris lumbricoides, Trichuris trichiura, Enterobius vermicularus, Strongyloides stercorals or Wuchereria bancrofti.

Preferably said protein is derived from T. colubriformis or H. contortus.

A particularly preferred protein of the invention is a protein having the amino sequence depicted in Table 3 or Table 8 or all, part, an analogue, homologue or derivative or combination thereof capable of conferring protective immunity against infection by a parasitic nematode.

In a further embodiment the invention provides a first nucleotide sequence encoding amino acid sequences of all, part, analogues, homologues, derivatives or combinations thereof of a protein capable of providing immunity to infection by a pathogenic nematode, a nucleotide sequence which hybridizes to said first nucleotide sequence, or a nucleotide sequence related by mutation including single and multiple base substitutions, insertions, and deletions to said first nucleotide sequence.

Preferred nucleotide sequences of the invention include those encoding the amino acid sequences depicted in Tables 1, 2, 3, 7 and 8, Particularly preferred DNA sequences of the invention are described in Tables 3, 7 and 8.

Preferred sequences in the invention are those encoding polypeptides corresponding to a 41 kD antigen. The DNA sequences embraced by the present invention can be prepared for example from T. colubriformis or H. contortus cells by extracting total DNA therefrom and isolating the sequences by standard techniques. Alternatively, the DNA may be prepared in vitro, synthetically or biosynthetically such as by the use of an mRNA template.

Also within the scope of the present invention is a process for selecting a DNA or RNA sequence coding for all, part, analogues, homologues, derivatives or combinations thereof of a protein according to the invention which process comprises providing one or more DNA or RNA sequences and determining which of said sequences hybridizes with a DNA or RNA sequence known to code for all, part, analogues, homologues, derivatives or combinations thereof of polypeptides having such an activity or providing an antiserum to said protein or parts thereof and identifying host-vector combinations that express said protein.

The above sequence may be from natural sources, may be RNA sequences, synthetic sequences, DNA sequences from recombinant DNA molecules or combinations of such sequences.

In a preferred form of the present invention, the process used to identify and characterize DNA coding for at least a portion of the protein involves the extraction of mRNA species from cells producing the protein, their conversion to double stranded DNA (cDNA) and the insertion of these into an autonomously replicating factor, such as a plasmid. This is followed by transformation of a host cell such as a bacterial strain with the factor and screening of the library produced with synthetic DNA probes which are complementary to the protein encoding mRNA or DNA sequences in order to detect those clones which contain DNA coding for the protein as opposed to any other cell proteinaceous components.

In a further embodiment the invention provides recombinant DNA molecules characterized by a DNA insert comprising a first DNA sequence which codes for amino acid sequences of all, part, analogues, homologues, derivatives or combinations thereof of a protein according to the invention, or a DNA sequence which hybridizes with said first sequence, said sequences being derived from whatever source including natural, synthetic, biosynthetic, or semi-synthetic sources and which sequences include those related by mutation, single or multiple base substitutions, deletions, insertions and inversions and including sequences which code for all, part, analogues, homologues, derivatives or combination thereof of a protein according to the invention, and vector DNA. Preferred recombinant DNA molecules of the invention include an expression control sequence operatively linked to said DNA insert. In one preferred form of the invention, said DNA insert is operatively linked to the β-galactosidase gene of E. coli. Other preferred control systems include those of the tryptophan (Trp operon), the leftward promoter of bacteriophage lambda (P_(L)) and hybrid promoter such as tac or viral promoters such as those of the long terminal repeat of Moloney leukaemia virus. A preferred recombinant DNA molecule of the present invention is a plasmid which contains a DNA insert as described above. Suitable plasmid vectors include the plasmids pUR290 and pUC18 and derivatives thereof. Preferred plasmids of the invention will be described in detail hereinafter and include pBTA593, pBTA597, pBTA598, pBTA702 & pBTA 704.

Alternatively, said recombinant DNA molecules may comprise said DNA insert linked to DNA of a suitable bacteriophage such as bacteriophage lambda or a derivative thereof.

The invention also provides a fused gene comprising a promoter, a translation start signal and a DNA sequence which is a DNA sequence according to the invention.

Also embraced within the present invention is a process for the manufacture of a recombinant DNA molecule which process comprises providing a DNA insert comprising a first DNA sequence which is a DNA sequence according to the invention and introducing said DNA insert into a cloning vector.

Preferably said DNA insert is introduced into the cloning vehicle in correct spacing and correct reading from with an expression control sequence.

In a further embodiment of the present invention there is provided a host transformed with at least one recombinant DNA molecule of the present invention and capable of expressing all, part, analogues, homologues, derivatives or combinations thereof of a protein according to the invention or a polypeptide having similar immunological or biological activity to said protective antigen. Suitable hosts include bacterial cells, yeasts, other fungi, vertebrate cells or insect cells, plant cells, human cells, human tissue cells or whole eukaryotic organisms. Suitable bacterial hosts include E. coli and other enteric organisms. Pseudomonas and Bacillus species. Preferred host cultures are identified as E. coli K12 derivatives. In particular JM109 and Y1090.

Particularly preferred transformant strains according to the invention are E. coli K-12 strains designated BTA 1621, BTA 1637, BTA 1638 and BTA 1684 and transformed with pBTA593, pBTA597, pBTA598 and pBTA702 respectively which were deposited with the American Type Culture Collection at 12301 Parklawn Drive Rockville, Md. 20852 USA on 17 Jun. 1987 and 28 Jun. 1988 for BTA 1684 and received accession numbers ATCC67438, ATCC67439, ATCC67440 and ATCC67738 respectively.

Also included within the scope of the present invention is a process for transforming a host which process comprises: providing a host and introducing into said host a recombinant DNA molecule of the present invention in correct reading frame.

The invention further provides expression products of the transformed host of the present invention which products comprise all, part, analogues, homologues, derivatives or combinations thereof of a protein according to the invention. Preferably these expression products are provided in substantially pure form.

In a preferred embodiment of the present invention the expression products comprise a first polypeptide sequence homologous to the host and a second polypeptide sequence which is the amino acid sequence coding for all, part, analogues, homologues, derivatives or combinations thereof of a protein according to the invention.

In a preferred embodiment of the present invention the first amino acid sequence is part or all of β-galactosidase and the host cell is E. coli.

In a further preferred embodiment of the invention the first sequence is the NH₂ -- terminal sequence of the expression product.

In a further embodiment of the present invention there is provided a process for the biosynthesis of a polypeptide which comprises all, part, analogues, homologues, derivatives or combinations thereof of a protein according to the invention which process comprises:

transforming a host with a recombinant DNA molecule of the present invention so that the host is capable of expressing a proteinaceous product which includes a polypeptide which is all, part, analogues, homologues, derivatives or combinations thereof of a protein according to the invention; culturing said host to obtain said expression; and collecting said polypeptide.

In a further embodiment the invention provides a vaccine comprising one or more expressed products or proteins of the invention together with a pharamaceutically acceptable carrier or diluent. Preferred vaccines include those suitable for oral administration or in injectable form and preferably include a pharmaceutically acceptable adjuvant.

In a further form the invention embraces antibody preparation prepared as a result of immunological challenge to a host by administration of one or more expression products, proteins or vaccines of the present invention. Such antibody preparations include polyclonal and monoclonal antibody preparations.

The invention also includes within its scope the epitope or the epitopes on the protein which are responsible for the protective immune response. These epitopes may be created artificially by the synthetic production of oligopeptides which contain sequences of portions of the protein which can be predicted from the results of immunochemical tests on fragments of the protein produced in bacteria or generated as a result of chemical or enzymatic cleavage of the native or recombinant peptides.

The invention also relates to antibodies generated against those epitopes and to antibodies generated against the variable region of those first antibodies, so called anti-idiotype antibodies, which mimic the protective epitopes of the protein and may be used as effective vaccines in either passive protection of animals (idiotypes) or active immunization of animals (anti-idiotypes) and thereby result in effective protection.

BEST METHOD OF CARRYING OUT THE INVENTION

The invention is further described with reference to the following Examples. It is to be understood that other methodologies analogous to those described below would be apparent to persons skilled in the art and these lie within the scope of the invention described.

EXAMPLE 1 Preparation of Extracts

Parasitological techniques--Third stage T. colubriformis larvae were obtained from culturing sheep faeces and Bearmanizing. There were kept in phosphate-buffered saline PBS (10 mM sodium phosphate, 150 mM sodium chloride, pH 7.4) at 4° C.

Extraction of T. colubriformis--2.0-2.5 g (centrifuged weight) of T. colubriformis third stage larvae were extracted 4 times each with 2.0 ml PBS plus 3.5 ml water. The buffer used in the first extraction contained in addition 1 mM phenylmethylsulphonyl fluoride, 0.2 mM p-chloromercuribenzoate, 5 mM ethylenediaminetetraacetate (EDTA) while the buffer used in the fourth extraction contained deoxyribonuclease (1 μg/1 ml). These extractions were followed by two extractions with PBS containing 1% sodium deoxychloate PBS-DOC! and 1 μg/ml of deoxyribonuclease. Extractions were performed using a motor-drive ground-glass Potter-Elvejhem homogenizer. During the PBS-DOC extractions, the samples were additionally treated for three one minute intervals in ice, with a Mullard ultrasonic disintegrator type 7685/2 at approximately 60 watts power. For gel electrophoresis (O'Donnell et al 1985) 20 μl of each centrifuged extract was dried and then heated for 3 min 100° C. with 15 μl sample buffer (Laemmli 1970). The PBS-DOC extract was centrifuged at 100,000 g for 1 hr before use and the pellet was discarded.

The SDS-PAGE patterns of successive PBS extractions of T. colubriformis third stage larvae followed by a PBS-DOC extract indicates that there are 4 major protein bands in the PBS-DOC extract which have been called 200 kD, 93 kD, 43 kD and 41 kD respectively. The 41 kD and 43 kD protein bands could be extracted using PBS without 1% DOC if the ultrasonic disintegrator was used but the 93 kD and 200 kD could not be efficiently extracted. The limited number of protein bands in the PBS-DOC extract contrasts with the multitude in the PBS extract. The 41 kD and 43 kD proteins could be efficiently extracted without the use of the ultrasonic disintegrator, with PBS-1% DOC but not with PBS-0.1% DOC. Ultrasonic disintegration gave a substantial increase in yield of proteins. If the deoxyribonuclease treatment was not performed, the PBS-DOC extract was very viscous due to the presence of DNA even after using the ultrasonic disintegrator.

EXAMPLE 2 Protein Analysis

Two-dimensional fractionation of the PBS-DOC extract

The procedure of Rubin and Leonardi (1983) was followed using Biolytes (Bio-Rad) for isoelectric focussing in the first dimension in 3 mm disc gels and Laemmli gels 1.5 mm thick and containing 10% polyacrylamide in the second dimension. The proteins were visualised with cold potassium chloride and the 41 kD protein was electro eluted and precipitated with cold methanol as described (Stearne et al 1985). This material was used for vaccination studies and for endoproteinase digestion.

Electrophoretic transfer form one- or two-dimensional gels to nitrocellulose (Bio-Rad) was performed in the standard manner. The nitrocellulose was then treated and probed with the various anti-sera by the procedure of Howe and Hershey (1981). Complexes were finally made visible by the reaction of bound antibodies with horseradish peroxidase conjugated to goat anti-rabbit IgG (Bio-Rad) followed, by 4-chloro-1-napthol and H₂ O₂.

Antisera from a rabbit immunised with 41 kD protein shows on immunoblots that the 41 kD protein exists in the 3rd and 4th larval stages as well as in the adults. The presence of carbohydrate was estimated in each component of the PBS-DOC extract following electroelution by assaying for the presence of glucosamine or galactosamine in acid hydrolysates and by the staining of gels using the method of Racusen (1979). Analyses showed no detectable carbohydrates in the 41 kD protein.

The purified 41 kD protein was reduced and carboxy methylated in 8M urea at pH 10.5 according to the procedure described by O'Donnell (1973). The protein was then digested with Armillaria mellea protease for 16 hrs at 37° C. in 50 mM ammonium hydrogen carbonate at a pH of approximately 8.0 and using an enzyme protein ratio of 1:50. The reaction mixture was then dried on a Savant Speed Vac Concentrator.

Oligopeptides resulting from protease digests of 41 kD were separated by HPLC using a Vydac C18 column and initial and final buffers consisting of 0.1 trifluoroacetic acid and 0.1% trifluoroacetic acid in 70% acetonitrile respectively.

Peptides isolated from the HPLC separation were sequenced manually using the procedure of McKern et al (1985) or automatically using a gas phase sequencer. The PTH amino acids were identified by HPLC.

Some of the amino acid sequences of HPLC-isolated peptides from an A. mellea digest of reduced and carboxymethylated 41 kD are presented in Table 1.

                                      TABLE 1                                      __________________________________________________________________________     Some amino acid sequences obtained from peptides produced following            digestion of the T. colubriformis 41kD protein with Armillaria mella           protease.                                                                      __________________________________________________________________________       LYS                                                                               VAL                                                                               MET                                                                               GLU                                                                               ASN                                                                               ARG                                                                               SER                                                                               PHE                                                                               GLN                                                                               ASP                                                                               GLU                                                                               GLU                                                                               ARG                                        LYS                                                                               MET                                                                               MET                                                                               GLN                                                                               THR                                                                               GLU                                                                               ASN                                                                               ASP                                                                               LEU                                                    LYS                                                                               ALA                                                                               ILE                                                                               SER                                                                               GLU                                                                               GLU                                                                               LEU                                                                               ASP                                                                               SER                                                                               ARG                                                                               PHE                                                                               GLN                                                                               GLU                                                                               LEU                                     LYS                                                                               GLU                                                                               VAL                                                                               ASP                                                                               ARG                                                                               LEU                                                                               GLU                                                                               ASP                                                                               GLU                                                                               LEU                                                                               VAL                                                                               HIS                                           LYS                                                                               TYR                                                                               ASP                                                                               GLU                                                                               VAL                                                                               ALA                                                                               ARG                                                          LYS                                                                               SER                                                                               LEU                                                                               GLU                                                                               VAL                                                                               SER                                                                               GLU                                                                               GLU                                                                               LYS                                                                               ALA                                                                               LEU                                                                               GLN                                                                               ARG                                                                               GLU                                     LYS                                                                               LEU                                                                               GLU                                                                               ARG                                                                               VAL                                                              No end group could be found on the undigested 41kD protein by the gas          phase                                                                          sequencer and it must therefore be assumed that it is blocked.                 __________________________________________________________________________

EXAMPLE 3 Identification and Characterization of the cDNA clone corresponding to the 41 kD protein of T. colibriformis

I. Construction of cDNA library

Messenger RNA was isolated from fourth stage larvae of T. colubriformis by grinding the larvae in a buffer containing 6M guanidine hydrochloride, 0.2M sodium acetate and 50 mM β-mercaptoethanol, followed by precipitation with ethanol and fractionation on an oligo(dT)-cellulose column. The mRNA was used as the template for synthesis of double-stranded cDNA using the Amersham ribonulease H/DNA polymerase I kit (Amersham cDNA synthesis system, #RPN, 1256) as recommended by the manufacturers. Following the addition of EcoRI linkers, the double-stranded cDNA was ligated to lambda gtll and packaged into viable bacteriophage which were used to infect Y1090 cells, essentially as described by Huynh et al (1985). Using the above methods, a cDNA library was established consisting of 2×10⁵ independent recombinants. The library was amplified and aliquots were screened using a synthetic oligonucleotide probe and duplicate filter lifts as described by Wallace et al (1985) and Benton and Davis (1977).

II. Hybridization Probe

The 23-mer oligonucleotide sequence used to screen the library was based on a peptide sequence obtained from amino acid sequence 2 (Table 1) of the 41 kD protein.

    ______________________________________                                         Peptide     LYS.MET.MET.GLN.THR.GLU.ASN.ASP                                    Oligonucleotide                                                                            5' AAA ATG ATG CAA ACT GAA AAT GA                                               G        G     C      G   C                                       Reverse Complement                                                                         5' TC ATT TTC AGT TTG CAT CAT TTT                                               G   C     G     C         C                                       ______________________________________                                    

The reverse complement sequence was synthesised and used for the screening.

(The codons used for threonine codons are based on the preferred codon usage of C. elegans: Maruyama, et al 1986)

Approximately 2×10⁵ recombinant bacteriophage were screened and 48 positives were detected.

III. DNA Sequencing

One of the selected clones, lambda gtll-4-41-6, contained a 700 bp insert which could be resected with EcoRI and subcloned into M13mp11 digested with the same enzyme. The DNA sequence of the subcloned insert was determined using the method of Sanger et al (1980) (Table 2).

A comparison of the sequence obtained with those in protein data banks shows that the 41 kD protein has homology with the amino sequences of tropomyosin from a variety of organisms e.g. the homology with rabbit was approximately 70%.

                                      TABLE 2                                      __________________________________________________________________________     DNA sequence of the Eco R1 cDNA fragment contained in plasmid pBTA 593         in                                                                             clone BTA 1621 and the translated amino acid sequence coding for part of       the 41kD protein of T. colubriformis.                                          __________________________________________________________________________      ##STR1##                                                                       ##STR2##                                                                       ##STR3##                                                                       ##STR4##                                                                       ##STR5##                                                                       ##STR6##                                                                       ##STR7##                                                                       ##STR8##                                                                       ##STR9##                                                                       ##STR10##                                                                      ##STR11##                                                                      ##STR12##                                                                      ##STR13##                                                                     __________________________________________________________________________

The 700 bp insert was used to screen further cDNA libraries and clones were isolated which contained more of the coding and non coding regions of the T. colubriformis 41 kD gene. The DNA sequence of these clones was determined and the DNA sequence and translated amino acid sequence for the 41 kD gene is shown in Table 3.

                                      TABLE 3                                      __________________________________________________________________________     Sequence of T. colubriformis (41kD antigen)                                    __________________________________________________________________________     1                             31                                               ATG                                                                               GAC                                                                               GCC                                                                               ATC                                                                               AAG                                                                               AAG                                                                               AAG                                                                               ATG                                                                               CAG                                                                               GCG                                                                               ATG                                                                               AAG                                                                               ATC                                                                               GAG                                                                               AAG                                                                               GAC                                                                               AAT                                                                               GCT                         Met                                                                               Asp                                                                               Ala                                                                               Ile                                                                               Lys                                                                               Lys                                                                               Lys                                                                               Met                                                                               Gln                                                                               Ala                                                                               Met                                                                               Lys                                                                               Ile                                                                               Glu                                                                               Lys                                                                               Asp                                                                               Asn                                                                               Ala                               61                            91                                         CTC                                                                               GAT                                                                               CGA                                                                               GCC                                                                               GAT                                                                               GCC                                                                               GCC                                                                               GAA                                                                               GAG                                                                               AAA                                                                               GTC                                                                               CGT                                                                               CAA                                                                               ATT                                                                               ACC                                                                               GAA                                                                               AAG                                                                               TTG                         Leu                                                                               Asp                                                                               Arg                                                                               Ala                                                                               Asp                                                                               Ala                                                                               Ala                                                                               Glu                                                                               Glu                                                                               Lys                                                                               Val                                                                               Arg                                                                               Gln                                                                               Ile                                                                               Thr                                                                               Glu                                                                               Lys                                                                               Leu                                  121                           151                                     GAG                                                                               CGA                                                                               GTT                                                                               GAA                                                                               GAA                                                                               GAG                                                                               CTC                                                                               CGT                                                                               GAC                                                                               ACA                                                                               CAA                                                                               AAG                                                                               AAA                                                                               ATG                                                                               ATG                                                                               CAA                                                                               ACA                                                                               GAA                         Glu                                                                               Arg                                                                               Val                                                                               Glu                                                                               Glu                                                                               Glu                                                                               Leu                                                                               Arg                                                                               Asp                                                                               Thr                                                                               Gln                                                                               Lys                                                                               Lys                                                                               Met                                                                               Met                                                                               Gln                                                                               Thr                                                                               Glu                                           181                           211                            AAC                                                                               GAT                                                                               TTG                                                                               GAC                                                                               AAG                                                                               GCT                                                                               CAG                                                                               GAA                                                                               GAT                                                                               TTG                                                                               GCT                                                                               GCA                                                                               GCC                                                                               ACC                                                                               AGC                                                                               CAG                                                                               TTG                                                                               GAA                         Asn                                                                               Asp                                                                               Leu                                                                               Asp                                                                               Lys                                                                               Ala                                                                               Gln                                                                               Glu                                                                               Asp                                                                               Leu                                                                               Ala                                                                               Ala                                                                               Ala                                                                               Thr                                                                               Ser                                                                               Gln                                                                               Leu                                                                               Glu                                                 241                                                    GAG                                                                               AAA                                                                               GAG                                                                               AAG                                                                               AAA                                                                               GTG                                                                               CAA                                                                               GAG                                                                               GCT                                                                               GAG                                                                               GCA                                                                               GAG                                                                               GTA                                                                               GCT                                                                               GCC                                                                               CTG                                                                               AAC                                                                               CGT                         Glu                                                                               Lys                                                                               Glu                                                                               Lys                                                                               Lys                                                                               Val                                                                               Gln                                                                               Glu                                                                               Ala                                                                               Glu                                                                               Ala                                                                               Glu                                                                               Val                                                                               Ala                                                                               Ala                                                                               Leu                                                                               Asn                                                                               Arg                         271                           301                                              CGC                                                                               ATG                                                                               ACT                                                                               CTT                                                                               CTC                                                                               GAA                                                                               GAA                                                                               GAG                                                                               CTT                                                                               GAA                                                                               CGT                                                                               GCT                                                                               GAA                                                                               GAA                                                                               CGT                                                                               TTG                                                                               AAG                                                                               ATC                         Arg                                                                               Met                                                                               Thr                                                                               Leu                                                                               Leu                                                                               Glu                                                                               Glu                                                                               Glu                                                                               Leu                                                                               Glu                                                                               Arg                                                                               Ala                                                                               Glu                                                                               Glu                                                                               Arg                                                                               Leu                                                                               Lys                                                                               Ile                               31                            361                                        GCC                                                                               ACT                                                                               GAA                                                                               AAA                                                                               CTC                                                                               GAA                                                                               GAG                                                                               GCC                                                                               ACT                                                                               CAC                                                                               AAT                                                                               GTC                                                                               GAC                                                                               GAG                                                                               TCC                                                                               GAG                                                                               CGT                                                                               GTA                         Ala                                                                               Thr                                                                               Glu                                                                               Lys                                                                               Leu                                                                               Glu                                                                               Glu                                                                               Ala                                                                               Thr                                                                               His                                                                               Asn                                                                               Val                                                                               Asp                                                                               Glu                                                                               Ser                                                                               Glu                                                                               Arg                                                                               Val                                     391                           421                                  CGC                                                                               AAA                                                                               GTG                                                                               ATG                                                                               GAG                                                                               AAC                                                                               GGC                                                                               TCA                                                                               TTC                                                                               CAA                                                                               GAT                                                                               GAG                                                                               GAG                                                                               CGT                                                                               GCC                                                                               AAC                                                                               ACG                                                                               ATT                         Arg                                                                               Lys                                                                               Val                                                                               Met                                                                               Glu                                                                               Asn                                                                               Gly                                                                               Ser                                                                               Phe                                                                               Gln                                                                               Asp                                                                               Glu                                                                               Glu                                                                               Arg                                                                               Ala                                                                               Asn                                                                               Thr                                                                               Ile                                           451                           481                            GAA                                                                               GCT                                                                               CAA                                                                               CTG                                                                               AAG                                                                               GAA                                                                               GCC                                                                               CAG                                                                               ATG                                                                               CTT                                                                               GCA                                                                               GAG                                                                               GAA                                                                               GCC                                                                               GAC                                                                               CGA                                                                               AAA                                                                               TAC                         Glu                                                                               Ala                                                                               Gln                                                                               Leu                                                                               Lys                                                                               Glu                                                                               Ala                                                                               Gln                                                                               Met                                                                               Leu                                                                               Ala                                                                               Glu                                                                               Glu                                                                               Asl                                                                               Asp                                                                               Arg                                                                               Lys                                                                               Tyr                                                 511                                                    GAT                                                                               GAG                                                                               GTC                                                                               GCC                                                                               CGT                                                                               AAA                                                                               TTG                                                                               GCC                                                                               ATG                                                                               GTT                                                                               GAA                                                                               GCT                                                                               GAT                                                                               CTT                                                                               GAA                                                                               AGG                                                                               GCC                                                                               GAA                         Asp                                                                               Glu                                                                               Val                                                                               Ala                                                                               Arg                                                                               Lys                                                                               Leu                                                                               Ala                                                                               Met                                                                               Val                                                                               Glu                                                                               Ala                                                                               Asp                                                                               Leu                                                                               Glu                                                                               Arg                                                                               Ala                                                                               Glu                         541                           571                                              GAA                                                                               CGT                                                                               GCT                                                                               GAA                                                                               GCC                                                                               GGA                                                                               GAG                                                                               AAC                                                                               AAG                                                                               ATC                                                                               GTC                                                                               GAA                                                                               TTG                                                                               GAA                                                                               GAG                                                                               GAA                                                                               TTG                                                                               CGT                         Glu                                                                               Arg                                                                               Ala                                                                               Glu                                                                               Ala                                                                               Gly                                                                               Glu                                                                               Asn                                                                               Lys                                                                               Ile                                                                               Val                                                                               Glu                                                                               Leu                                                                               Glu                                                                               Glu                                                                               Glu                                                                               Leu                                                                               Arg                               601                           631                                        GTC                                                                               GTC                                                                               GGA                                                                               AAT                                                                               AAC                                                                               CTG                                                                               AAA                                                                               TCA                                                                               CTC                                                                               GAA                                                                               GTG                                                                               TCC                                                                               GAA                                                                               GAA                                                                               AAG                                                                               GCA                                                                               CTG                                                                               CAA                         Val                                                                               Val                                                                               Gly                                                                               Asn                                                                               Asn                                                                               Leu                                                                               Lys                                                                               Ser                                                                               Leu                                                                               Glu                                                                               Val                                                                               Ser                                                                               Glu                                                                               Glu                                                                               Lys                                                                               Ala                                                                               Leu                                                                               Gln                                     661                           691                                  CGT                                                                               GAG                                                                               GAC                                                                               TCA                                                                               TAC                                                                               GAA                                                                               GAG                                                                               CAG                                                                               ATT                                                                               CGT                                                                               ACC                                                                               ATT                                                                               TCA                                                                               TCT                                                                               CGT                                                                               CTG                                                                               AAG                                                                               GAG                         Arg                                                                               Glu                                                                               Asp                                                                               Ser                                                                               Tyr                                                                               Glu                                                                               Glu                                                                               Gln                                                                               Ile                                                                               Arg                                                                               Thr                                                                               Ile                                                                               Ser                                                                               Ser                                                                               Arg                                                                               Leu                                                                               Lys                                                                               Glu                                           721                           751                            GCG                                                                               GAA                                                                               ACC                                                                               CGT                                                                               GCT                                                                               GAA                                                                               TTC                                                                               GCC                                                                               GAG                                                                               CGC                                                                               TCC                                                                               GTG                                                                               CAG                                                                               AAG                                                                               CTC                                                                               CAG                                                                               AAG                                                                               GAA                         Ala                                                                               Glu                                                                               Thr                                                                               Arg                                                                               Ala                                                                               Glu                                                                               Phe                                                                               Ala                                                                               Glu                                                                               Arg                                                                               Ser                                                                               Val                                                                               Gln                                                                               Lys                                                                               Leu                                                                               Gln                                                                               Lys                                                                               Glu                                                 781                                                    GTC                                                                               GAC                                                                               AGA                                                                               CTC                                                                               GAG                                                                               GAT                                                                               GAA                                                                               TTG                                                                               GTA                                                                               CAT                                                                               GAG                                                                               AAG                                                                               GAG                                                                               AGA                                                                               TAC                                                                               AAG                                                                               GCG                                                                               ATT                         Val                                                                               Asp                                                                               Arg                                                                               Leu                                                                               Glu                                                                               Asp                                                                               Glu                                                                               Leu                                                                               Val                                                                               His                                                                               Glu                                                                               Lys                                                                               Glu                                                                               Arg                                                                               Tyr                                                                               Lys                                                                               Ala                                                                               Ile                         811                           841                                              TCC                                                                               GAG                                                                               GAG                                                                               CTT                                                                               GAC                                                                               TCG                                                                               ACC                                                                               TTC                                                                               CAA                                                                               GAA                                                                               CTC                                                                               TCC                                                                               GGC                                                                               TAT                                     Ser                                                                               Glu                                                                               Glu                                                                               Leu                                                                               Asp                                                                               Ser                                                                               Thr                                                                               Phe                                                                               Gln                                                                               Glu                                                                               Leu                                                                               Ser                                                                               Gly                                                                               Tyr                                     __________________________________________________________________________

Production of the 41 kD protein as a Recombinant Fusion Protein with E. coli β-galactosidase.

IV. Subcloning the 700 bp 41 kD cDNA fragment into pUR290

A. Preparation of fragment.

DNA was prepared from clone lambdagtL4/41/6 according to Maniatis et al (1982). The DNA was cut with the restriction enzyme EcoRl to release the 700 bp insert which was then purified for subsequent subcloning. Purification was carried out as follows: the EcoRl-cut DNA was separated by electrophoresis in an agarose gel and the 700 bp fragment collected on and eluted from NA45 paper according to the manufacturer's instructions (Schleicher & Schuell). The 700 bp DNA was then precipitated with isopropanol and redissolved in TE (10 mM Tris pH7.5-1 mM EDTA).

B. Cloning into pUR290;

The expression vector pUR290 Ruther and Muller-Hill, 1983! is a plasmid containing the bacterial β-galactosidase gene with a number of restriction enzyme sites near the 3' end into which foreign sequences can be inserted. The purified 700 bp cDNA fragment was ligated with EcoRl digested pUR290 using methods described by Maniatis et al (1982). Coincidentally, the reading frame of the β-galactosidase gene at the EcoRl restriction enzyme site is in phase with that of the 41 kD 700 bp cDNA fragment.

V. Detection of pUR clones producing fusion protein

The pUR clones expressing 41 kD antigen were identified using serum from a rabbit which had been vaccinated with purified 41 kD protein. The colonies were grown on agar containing ampilicillin and IPTG and replicas of the colonies were made on nitrocellulose. Those colonies expressing 41 kD antigen were identified according to standard immunological screening methods Maniatis et al (1982)! and probed with antisera-peroxidase method. A number of positive colonies were picked and grown in liquid culture for analysis on SDS-PAGE (FIG. 3).

VI. Size of fusion protein on IDS-PAGE

The recombinant β-galactosidase/41 kD protein produced by strain BTA 1621 (which contains plasmid pBTA 593) is about 140 kD MW, of which approximately 27 kD is T. colubriformis in origin. Unfused β-galactosidase is about 120 kD MW.

VII. Growth of BTA 1621 in liquid culture+IPTG

Recombinant clone BTA 1621, containing the 41 kD cDNA fragment inserted, in frame, at the 3' end of the bacterial β-galactosidase gene of pUR290 (plasmid pBTA 593), was grown in LB bacterial culture medium+ampicillin+IPGT at 21° overnight Maniatis et al (1982!. The bacteria were then collected and further processed for protein purification.

VIII. Purification of fusion protein for animal testing

For purification of the β-galactosidase/41 kD fusion protein, clone BTA 1621 was grown overnight in 500 ml LB+Amp(50 ug/ml)+IPTG(1 μg/ml) at 20° C. The bacteria were collected by centrifugation, treated with lysozyme (500 ug/ml in Tris EDTA buffer, 5' at 4°) and lysed by addition of NP40 to 1% v/v. Deoxyribonuclease was added to 100 ug/ml, MgCl₂ to 50 mM and the lysate incubated at 4° overnight. The lysate was then centrifuged at 10,000 rpm in a Beckman JA20 rotor for 10 min. The pellet, containing most of the fusion protein, was dissolved in 8M urea/1% β-mercaptoethanol in 50 mM Tris-HCl(pH8), 100 mM NaCl and then chromatographed on a Superose 6 column (1.6 cm×80 cm). Using SDS-PAGE, fractions containing the fusion protein were identified and then pooled. Western blots Towbin H, et al! indicate a positive reaction between the rabbit anti 41 kD antibody and the purified protein (see FIG. 3).

EXAMPLE 4 Experimental animals were chosen as described previously (O'Donnell et al, 1985)

41 kD was purified from PBS-DOC extracts of third state larvae of Trichostrongylus colubriformis, separated by two dimensional SDS-PAGE and electroeluted. This material was used to vaccinate guinea pigs intraperitoneally (O'Donnell et al 1985). It can be seen (Table 4) that the purified 41 kD fraction gave substantial protection (ranging from 43-51% against T. colubriformis challenge infection in guinea pigs following vaccination.

                                      TABLE 4                                      __________________________________________________________________________     Vaccination of guinea pigs with purified 41kD protein from                     T. colubriformis.                                                                     Experiment No.                                                          Worm counts                                                                           1        2        3a       3b                                           __________________________________________________________________________     Controsl                                                                              671 ± 257 (n = 10)                                                                   963 ± 206 (n = 10)                                                                   1056 ± 353 (n = 10)                                                                  1056 ± 353 (n = 10)                       Vaccinates                                                                            364 ± 159 (n = 8)                                                                    549 ± 351 (n = 6)                                                                     550 ± 148 (n = 5)                                                                    513 ± 149 (n = 5)                        % Protection                                                                           46       43       48       51                                          μg injected                                                                         85       50       50       100                                         __________________________________________________________________________

All animals were injected intraperitoneally with the T. colubriformis 41 kD protein isolated from third stage larvae. The animals were challenged with 2000 infective larvae 28 days later and killed for worm counts 13 days post challenge.

Protection of 43-51% was given by the 41 kD immunogenic fraction when eluted from a Laemmli gel. It is reasonable to expect that it may be more potent if prepared by non denaturing means.

This 41 kD fraction exists in all juvenile and adult stages as demonstrated by immunoblotting with rabbit antisera against material electro-eluted after SDS-PAGE. However, antibodies (IgA, IgG or IgM) against 41 kD protein could not be detected in sera from several naturally-infected guinea pigs and sheep nor in the mesenteric lymph from a sheep infected with T. colubriformis (as studied by the immunoblot technique).

Use of 41 kD Fusion Protein as a VACCINE

As shown in Table 5 intraperitoneal injection of the recombinant 41 kD fusion protein purified from BTA 1621 gave similar protection to that achieved by the original 41 kD protein extracted from T. colubriformis larvae.

                  TABLE 5                                                          ______________________________________                                         VACCINATION OF GUINEA PIGS WITH 41kD - FUSION                                  PROTEIN DERIVED FROM CLONE BTA 1621                                            (Worm Numbers)                                                                 Controls         Vaccinates   % Protection                                     ______________________________________                                         Mean 1135 ±  263! (n = 12)                                                                       416 ±  220! (n = 3)                                                                      63%                                          ______________________________________                                          T-test significance <0.001                                               

Animals were vaccinated with 450 μg fusion protein per guinea pig, challenged with 2000 infective T. colubriformis larvae 21 days later. Animals were killed for worm counts 13 days post-challenge.

EXAMPLE 5 Isolation and Characterisation of a cDNA Clone Encoding the Haemonchus Contortus 41 kD Protein

Polyadenylated mRNA was extracted from young adult H. contortus and a cDNA library was constructed in lambda gtll using the methods essentially as described in International Patent Application No. PCT/AU87/00401.

Approximately 3×10⁴ recombinant bacteriophage were screened with DNA fragments isolated from a clone coding for the T. colubriformis 41 kD protein. Duplicate nitrocellulose filters were prepared as described by Maniatis 1982. The probe was labelled according to the method of Feinberg and Vogelstein 1984. Hybridisation conditions used were as described by Reed. Four positive clones were detected and purified.

One of the isolated clones, 41/4, contained approximately 1.38 kb of H. contortus cDNA with an internal Eco RI site. Digestion of the DNA isolated from the clone with Eco RI produced cDNA fragments of approximately 900 and 480 bp.

DNA was prepared from clone 41/4 (Maniatis 1982) and digested with Eco RI under conditions leading to partial or complete digestion. The DNA was electrophorsed in agarose gels and the 480 bp, 900 bp and 1.38 Kb fragments were isolated using NA45 paper (Schleicher and Schuell). The 480 and 900 bp fragments were subcloned into a vector pBTA224 which is a derivative of pUR290 (Ruther and Miller-Hill 1982) from which the Eco RI site outside the β-galactosidase gene has been removed. The 1.38 Kb partial digest fragment was subcloned into pBTA502 which is a derivative of pUC18 (Yamisch-Perron, Vierira and Messing (1985)). Three clones were isolated and will be described:

Clone BTA1637 contains plasmid pBTA597 which is the 480 bp fragment in pBTA224: clone BTA1638 contains plasmid pBTA598 which is the 900 bp fragment in pBTA224: cone BTA1684 contains plasmid pBTA702 which is the 1.38 Kb partial digest fragment in plasmid pBTA502 (host strain is JM109 in all cases).

A series of deletions of the 1.38 Kb fragment contained in pBTA702 were made by digesting samples of the DNA with BamHI and Pstl followed by digestion of the linearized DNA with econuclease III and mung bean nuclease, for various periods of time, and religating the reaction products. The DNA was then transformed into JM109 and ampicillin resistant colonies were picked. The plasmids were characterized and the DNA sequence of the inserts in a selection of those transformants was determined using the method of Sanger et al as adapted by Hattori and Sakaki (1986) for use with double stranded DNA templates. The complete sequence of the cDNA segment in pBTA702 (Table 6) shows extensive homology to the cloned T. colubriformis gene.

                                      TABLE 6                                      __________________________________________________________________________     Sequence of the cDNA insert in pBTA702.                                        __________________________________________________________________________     10      20      30      40      50      60      70                             GATTCGGAGC                                                                             GGCCACAGAC                                                                             GAAAGCAGCT                                                                             CGGCGTTTTT                                                                             CGGTCGTCGC                                                                             TTCCCGTTTT                                                                             GTTTGATCTC                     80      90      100     110     120     130     140                            TTCGAGAGGA                                                                             GCAGAAGAGA                                                                             GCGGTGTGAG                                                                             CGACTGCCGC                                                                             CCTCACAATT                                                                             TGCTGACAGT                                                                             CGCGCGACTT                     150     160     170     180     190     200     210                            CCACAAGTGC                                                                             TTCCACATTT                                                                             CTTGCTTGTC                                                                             TCGCTACGGC                                                                             TACCGATTTG                                                                             TTCTAAAGTA                                                                             TCATTCACAT                     220     230     240     250     260     270     280                            CTGAATCCAC                                                                             TACTGCCATC                                                                             ATGTCGAAAG                                                                             TGAACAAAGA                                                                             AGGAGCTCAG                                                                             CAGACATCTC                                                                             TGCTCGATGT                     290     300     310     320     330     340     350                            CCTCAAGAAG                                                                             AAAATGCGCC                                                                             AAGCCCGCGA                                                                             AGAGGCTGAA                                                                             GCTGCAAAGG                                                                             ACGAAGCCGA                                                                             TGAAGTGCAA                     360     370     380     390     400     410     420                            CGACAGCTCG                                                                             AAGAGGAACG                                                                             TAAAAAGCGT                                                                             GAGGACGCTG                                                                             AAGCTGAAGT                                                                             GGCAGCATTG                                                                             AATCGCCGCA                     430     440     450     460     470     480     490                            TTGTATTGGT                                                                             TGAGGAGGAT                                                                             TTGGAACGTA                                                                             CTGAAGATCG                                                                             TCTGAAGATT                                                                             GCCACATCCA                                                                             AATTGGAAGA                     500     510     520     530     540     550     560                            GGCTTCGAAG                                                                             GCAGCCGATG                                                                             AGGCTGAACG                                                                             AGCTCGAAGA                                                                             TCGTTGGAAA                                                                             ATCGTGTCGA                                                                             TGTCGATGAG                     570     580     590     600     610     620     630                            GATCGTTGTG                                                                             CCGAGCTCGA                                                                             AACGAAACTA                                                                             CGTGAAGCTC                                                                             AAGCTCTTCT                                                                             GCATGAAACA                                                                             GAGAGTAAGA                     640     650     660     670     680     690     700                            GCGAAGAGGT                                                                             CGCCCGTAAG                                                                             CTGGCTATGG                                                                             TTGAAGCTGA                                                                             TCTCGAAAGA                                                                             GCCGAAGAAC                                                                             GTGCTGAAGC                     710     720     730     740     750     760     770                            CGGAGAGAAC                                                                             AAGATCGTCG                                                                             AGTTGGAAGA                                                                             GGAACTTCGT                                                                             GTCGTCGGAA                                                                             ACAACTTGAA                                                                             GTCACTTGAG                     780     790     800     810     820     830     840                            GTGTCTGAAG                                                                             AAAAGGCCCT                                                                             TCAACGTGAA                                                                             GACTCATACG                                                                             AGGAACAGAT                                                                             TCGTACTATC                                                                             TCAGCTCGTC                     850     860     870     880     890     900     910                            TGAAGGAGGC                                                                             GGAAACCCGT                                                                             GCCGAATTCG                                                                             CTGAGCGTTC                                                                             CGTGCAGAAA                                                                             CTCCAGAAGG                                                                             AAGTCGACAG                     920     930     940     950     960     970     980                            ACTGGAGGAT                                                                             GAATTGGTAC                                                                             ATGAGAAGGA                                                                             GAGATACAAG                                                                             GCAATTTCCG                                                                             AGGAGCTTGA                                                                             CTCGACCTTC                     990     1000    1010    1020    1030    1040    1050                           CAAGAACTGT                                                                             CCGGCTATTG                                                                             ATTTCTTCAT                                                                             TTTTTCCATC                                                                             AATCCATCCA                                                                             AATCATCCCC                                                                             GTCATTGCTG                     1060    1070    1080    1090    1100    1110    1120                           ACATTTTTC                                                                              TACCGTACAT                                                                             GTGTCTCTTT                                                                             CCTTCTGTGC                                                                             TCCTGTTTCA                                                                             TCTATCATTC                                                                             GCAGTTTGTA                     1130    1140    1150    1160    1170    1180    1190                           GTCTACGCAA                                                                             TCACTCGAAC                                                                             GGTAATGCAT                                                                             CGCCTGAATA                                                                             TGTTCATAAT                                                                             CCCCCTCCGT                                                                             TTTAGCTTCT                     1200    1210    1220    1230    1240    1250    1260                           CATAGCACAT                                                                             TCGAGAAGCT                                                                             TCGCTTCACT                                                                             CTACCCAGTT                                                                             CTGTTTTACT                                                                             AACATCTATT                                                                             CGCGTCTTTT                     1270    1280    1290    1300    1310    1320    1330                           CACCTTCTTT                                                                             TTTGATGAAG                                                                             AGCAGGTCAA                                                                             AATAAAGAAT                                                                             TGAACGAAAA                                                                             AAAAAAAAAA                                                                             AAAAAAA                        __________________________________________________________________________

From the DNA sequence, an open reading frame of 1032 base pairs can be identified which codes for a protein containing 344 amino acids (Table 6): The amino acid sequence corresponding to some of the peptide fragments generated by A. millea digestion of the T. colubriformis antigen can be identified in the H. contortus cloned sequence (underlined in Table 7). The DNA sequence homology between the H. contortus and T. colubriformis clones it high over the region from amino acids 130 to 344. The N-terminal 130 amino acids however contain no such homology. Further analysis of the DNA sequence shows that the N-terminal segment of the H. contortus sequence is derived from DNA coding for 10 amino acids of β-galactosidase followed by 77 irrelevant amino acids and a segment of 43 amino acids extending from a putative initiation codon. The DNA is fortuitously in phase and so gives rise to a 344 amino acid continuous open reading frame. In support of this proposal, the ATG which is presumed to be the initiating codon for the H. contortus antigen is preceded by a sequence TGCCATC which closely resembles the consensus sequence for a Shine-Dalgarno sequence in vertebrate genes vis agcCAcC.

                                      TABLE 7                                      __________________________________________________________________________     Sequence of the fusion protein produced by strain BTA 1684.                    __________________________________________________________________________     1                             31                                               ATG                                                                               ATT                                                                               ACG                                                                               AAT                                                                               TGC                                                                               CCG                                                                               GGA                                                                               GAT                                                                               CTG                                                                               GAA                                                                               TTC                                                                               CGA                                                                               TTC                                                                               GGA                                                                               GCG                                                                               GCC                                                                               ACA                                                                               GAC                         Met                                                                               Ile                                                                               Thr                                                                               Asn                                                                               Cys                                                                               Pro                                                                               Gly                                                                               Asp                                                                               Leu                                                                               Glu                                                                               Phe                                                                               Arg                                                                               Phe                                                                               Gly                                                                               Ala                                                                               Ala                                                                               Thr                                                                               Asp                               61                            91                                         GAA                                                                               AGC                                                                               AGC                                                                               TCG                                                                               GCG                                                                               TTT                                                                               TTC                                                                               GGT                                                                               CGT                                                                               CGC                                                                               TTC                                                                               CCG                                                                               TTT                                                                               TGT                                                                               TTG                                                                               ATC                                                                               TCT                                                                               TCG                         Glu                                                                               Ser                                                                               Ser                                                                               Ser                                                                               Ala                                                                               Phe                                                                               Phe                                                                               Gly                                                                               Arg                                                                               Arg                                                                               Phe                                                                               Pro                                                                               Phe                                                                               Cys                                                                               Leu                                                                               Ile                                                                               Ser                                                                               Ser                                     121                           151                                  AGA                                                                               GGA                                                                               GCA                                                                               GAA                                                                               GAG                                                                               AGC                                                                               GGT                                                                               GTG                                                                               AGC                                                                               GAC                                                                               TGC                                                                               CGC                                                                               CCT                                                                               CAC                                                                               AAT                                                                               TTG                                                                               CTG                                                                               ACA                         Arg                                                                               Gly                                                                               Ala                                                                               Glu                                                                               Glu                                                                               Ser                                                                               Gly                                                                               Val                                                                               Ser                                                                               Asp                                                                               Cys                                                                               Arg                                                                               Pro                                                                               His                                                                               Asn                                                                               Leu                                                                               Leu                                                                               Thr                                           181                           211                            GTC                                                                               GCG                                                                               CGA                                                                               CTT                                                                               CCA                                                                               CAA                                                                               GTG                                                                               CTT                                                                               CCA                                                                               CAT                                                                               TTC                                                                               TTG                                                                               CTT                                                                               GTC                                                                               TCG                                                                               CTA                                                                               CGG                                                                               CTA                         Val                                                                               Ala                                                                               Arg                                                                               Leu                                                                               Pro                                                                               Gln                                                                               Val                                                                               Leu                                                                               Pro                                                                               His                                                                               Phe                                                                               Leu                                                                               Leu                                                                               Val                                                                               Ser                                                                               Leu                                                                               Arg                                                                               Leu                                                 241                                                    CCG                                                                               ATT                                                                               TGT                                                                               TCT                                                                               AAA                                                                               GTA                                                                               TCA                                                                               TTC                                                                               ACA                                                                               TCT                                                                               GAA                                                                               TCC                                                                               ACT                                                                               ACT                                                                               GCC                                                                               ATC                                                                               ATG                                                                               TCG                         Pro                                                                               Ile                                                                               Cys                                                                               Ser                                                                               Lys                                                                               Val                                                                               Ser                                                                               Phe                                                                               Thr                                                                               Ser                                                                               Glu                                                                               Ser                                                                               Thr                                                                               Thr                                                                               Ala                                                                               Ile                                                                               Met                                                                               Ser                         271                           301                                              AAA                                                                               GTG                                                                               AAC                                                                               AAA                                                                               GAA                                                                               GGA                                                                               GCT                                                                               CAG                                                                               CAG                                                                               ACA                                                                               TCT                                                                               CTG                                                                               CTC                                                                               GAT                                                                               GTC                                                                               CTC                                                                               AAG                                                                               AAG                         Lys                                                                               Val                                                                               Asn                                                                               Lys                                                                               Glu                                                                               Gly                                                                               Ala                                                                               Gln                                                                               Gln                                                                               Thr                                                                               Ser                                                                               Leu                                                                               Leu                                                                               Asp                                                                               Val                                                                               Leu                                                                               Lys                                                                               Lys                               331                           361                                        AAA                                                                               ATG                                                                               CGC                                                                               CAA                                                                               GCC                                                                               CGC                                                                               GAA                                                                               GAG                                                                               GCT                                                                               GAA                                                                               GCT                                                                               GCA                                                                               AAG                                                                               GAC                                                                               GAA                                                                               GCC                                                                               GAT                                                                               GAA                         Lys                                                                               Met                                                                               Arg                                                                               Gln                                                                               Ala                                                                               Arg                                                                               Glu                                                                               Glu                                                                               Ala                                                                               Glu                                                                               Ala                                                                               Ala                                                                               Lys                                                                               Asp                                                                               Glu                                                                               Ala                                                                               Asp                                                                               Glu                                     391                           421                                  GTG                                                                               CAA                                                                               CGA                                                                               CAG                                                                               CTC                                                                               GAA                                                                               GAG                                                                               GAA                                                                               CGT                                                                               AAA                                                                               AAG                                                                               CGT                                                                               GAG                                                                               GAC                                                                               GCT                                                                               GAA                                                                               GCT                                                                               GAA                         Val                                                                               Gln                                                                               Arg                                                                               Gln                                                                               Leu                                                                               Glu                                                                               Glu                                                                               Glu                                                                               Arg                                                                               Lys                                                                               Lys                                                                               Arg                                                                               Glu                                                                               Asp                                                                               Ala                                                                               Glu                                                                               Ala                                                                               Glu                                           451                           481                            GTG                                                                               GCA                                                                               GCA                                                                               TTG                                                                               AAT                                                                               CGC                                                                               CGC                                                                               ATT                                                                               GTA                                                                               TTG                                                                               GTT                                                                               GAG                                                                               GAG                                                                               GAT                                                                               TTG                                                                               GAA                                                                               CGT                                                                               ACT                         Val                                                                               Ala                                                                               Ala                                                                               Leu                                                                               Asn                                                                               Arg                                                                               Arg                                                                               Ile                                                                               Val                                                                               Leu                                                                               Val                                                                               Glu                                                                               Glu                                                                               Asp                                                                               Leu                                                                               Glu                                                                               Arg                                                                               Thr                                                 511                                                    GAA                                                                               GAT                                                                               CGT                                                                               CTG                                                                               AAG                                                                               ATT                                                                               GCC                                                                               ACA                                                                               TCC                                                                               AAA                                                                               TTG                                                                               GAA                                                                               GAG                                                                               GCT                                                                               TCG                                                                               AAG                                                                               GCA                                                                               GCC                         Glu                                                                               Asp                                                                               Arg                                                                               Leu                                                                               Lys                                                                               Ile                                                                               Ala                                                                               Thr                                                                               Ser                                                                               Lys                                                                               Leu                                                                               Glu                                                                               Glu                                                                               Ala                                                                               Ser                                                                               Lys                                                                               Ala                                                                               Ala                         541                           571                                              GAT                                                                               GAG                                                                               GCT                                                                               GAA                                                                               CGA                                                                               GCT                                                                               CGA                                                                               AGA                                                                               TCG                                                                               TTG                                                                               GAA                                                                               AAT                                                                               CGT                                                                               GTC                                                                               GAT                                                                               GTC                                                                               GAT                                                                               GAG                         Asp                                                                               Glu                                                                               Ala                                                                               Glu                                                                               Arg                                                                               Ala                                                                               Arg                                                                               Arg                                                                               Ser                                                                               Leu                                                                               Glu                                                                               Asn                                                                               Arg                                                                               Val                                                                               Asp                                                                               Val                                                                               Asp                                                                               Glu                               601                           631                                        GAT                                                                               CGT                                                                               TGT                                                                               GCC                                                                               GAG                                                                               CTC                                                                               GAA                                                                               ACG                                                                               AAA                                                                               CTA                                                                               CGT                                                                               GAA                                                                               GCT                                                                               CAA                                                                               GCT                                                                               CTT                                                                               CTG                                                                               CAT                         Asp                                                                               Arg                                                                               Cys                                                                               Ala                                                                               Glu                                                                               Leu                                                                               Glu                                                                               Thr                                                                               Lys                                                                               Leu                                                                               Arg                                                                               Glu                                                                               Ala                                                                               Gln                                                                               Ala                                                                               Leu                                                                               Leu                                                                               His                                     661                           691                                  GAA                                                                               ACA                                                                               GAG                                                                               AGT                                                                               AAG                                                                               AGC                                                                               GAA                                                                               GAG                                                                               GTC                                                                               GCC                                                                               CGT                                                                               AAG                                                                               CTG                                                                               GCT                                                                               ATG                                                                               GTT                                                                               GAA                                                                               GCT                         Glu                                                                               Thr                                                                               Glu                                                                               Ser                                                                               Lys                                                                               Ser                                                                               Glu                                                                               Glu                                                                               Val                                                                               Ala                                                                               Arg                                                                               Lys                                                                               Leu                                                                               Ala                                                                               Met                                                                               Val                                                                               Glu                                                                               Ala                                           721                           751                            GAT                                                                               CTC                                                                               GAA                                                                               AGA                                                                               GCC                                                                               GAA                                                                               GAA                                                                               CGT                                                                               GCT                                                                               GAA                                                                               GCC                                                                               GGA                                                                               GAG                                                                               AAC                                                                               AAG                                                                               ATC                                                                               GTC                                                                               GAG                         Asp                                                                               Leu                                                                               Glu                                                                               Arg                                                                               Ala                                                                               Glu                                                                               Glu                                                                               Arg                                                                               Ala                                                                               Glu                                                                               Ala                                                                               Gly                                                                               Glu                                                                               Asn                                                                               Lys                                                                               Ile                                                                               Val                                                                               Glu                                                 781                                                    TTG                                                                               GAA                                                                               GAG                                                                               GAA                                                                               CTT                                                                               CGT                                                                               GTC                                                                               GTC                                                                               GGA                                                                               AAC                                                                               AAC                                                                               TTG                                                                               AAG                                                                               TCA                                                                               CTT                                                                               GAG                                                                               GTG                                                                               TCT                         Leu                                                                               Glu                                                                               Glu                                                                               Glu                                                                               Leu                                                                               Arg                                                                               Val                                                                               Val                                                                               Gly                                                                               Asn                                                                               Asn                                                                               Leu                                                                               Lys                                                                               Ser                                                                               Leu                                                                               Glu                                                                               Val                                                                               Ser                         811                           841                                              GAA                                                                               GAA                                                                               AAG                                                                               GCC                                                                               CTT                                                                               CAA                                                                               CGT                                                                               GAA                                                                               GAC                                                                               TCA                                                                               TAC                                                                               GAG                                                                               GAA                                                                               CAG                                                                               ATT                                                                               CGT                                                                               ACT                                                                               ATC                         Glu                                                                               Glu                                                                               Lys                                                                               Ala                                                                               Leu                                                                               Gln                                                                               Arg                                                                               Glu                                                                               Asp                                                                               Ser                                                                               Tyr                                                                               Glu                                                                               Glu                                                                               Gln                                                                               Ile                                                                               Arg                                                                               Thr                                                                               Ile                               871                           901                                        TCA                                                                               GCT                                                                               CGT                                                                               CTG                                                                               AAG                                                                               GAG                                                                               GCG                                                                               GAA                                                                               ACC                                                                               CGT                                                                               GCC                                                                               GAA                                                                               TTC                                                                               GCT                                                                               GAG                                                                               CGT                                                                               TCC                                                                               GTG                         Ser                                                                               Ala                                                                               Arg                                                                               Leu                                                                               Lys                                                                               Glu                                                                               Ala                                                                               Glu                                                                               Thr                                                                               Arg                                                                               Ala                                                                               Glu                                                                               Phe                                                                               Ala                                                                               Glu                                                                               Arg                                                                               Ser                                                                               Val                                     931                           961                                  CAG                                                                               AAA                                                                               CTC                                                                               CAG                                                                               AAG                                                                               GAA                                                                               GTC                                                                               GAC                                                                               AGA                                                                               CTG                                                                               GAG                                                                               GAT                                                                               GAA                                                                               TTG                                                                               GTA                                                                               CAT                                                                               GAG                                                                               AAG                         Gln                                                                               Lys                                                                               Leu                                                                               Gln                                                                               Lys                                                                               Glu                                                                               Val                                                                               Asp                                                                               Arg                                                                               Leu                                                                               Glu                                                                               Asp                                                                               Glu                                                                               Leu                                                                               Val                                                                               His                                                                               Glu                                                                               Lys                                        991                           1021                              GAG                                                                               AGA                                                                               TAC                                                                               AAG                                                                               GCA                                                                               ATT                                                                               TCC                                                                               GAG                                                                               GAG                                                                               CTT                                                                               GAC                                                                               TCG                                                                               ACC                                                                               TTC                                                                               CAA                                                                               GAA                                                                               CTG                                                                               TCC                         Glu                                                                               Arg                                                                               Tyr                                                                               Lys                                                                               Ala                                                                               Ile                                                                               Ser                                                                               Glu                                                                               Glu                                                                               Leu                                                                               Asp                                                                               Ser                                                                               Thr                                                                               Phe                                                                               Gln                                                                               Glu                                                                               Leu                                                                               Ser                         GGC                                                                               TAT                                                                         Gly                                                                               Tyr                                                                         __________________________________________________________________________

Recombinant organisms have been constructed which lack the irrelevant N-terminal 87 amino acids, in order to produce a near native H. contortus antigen for vaccination studies. DNA from pBTA702 was digested with restriction enzymes Sma I and Asu II in combination and the 3514 and 513 bp fragments were purified. The 513 bp fragment was then digested with DdeI and the 238 bp fragment was purified. This 238 bp fragment was then ligated to the 3514 bp fragment, the non-cohesive end was rendered blunt by treatment with the Klenow fragment of DNA polymerase I and the DNA was then ligated to form circles and used to transform E. coli strain J109. Ampicillin resistant colonies were selected, the plasmid DNA was isolated and the DNA sequence was confirmed by restriction enzyme analysis. The DNA in plasmid pBTA 704 (strain BTA 1686) codes for a molecule of 252 amino acids (Table 8) which is only 4 amino acids shorter than mature H. contortus antigen. The 6 amino acids on the N-terminus are derived from β-galactosidase and the initiating methionine is preceded by the β-galactosidase Shine-Dalgarno sequence.

                                      TABLE 8                                      __________________________________________________________________________     Sequence of the fusion protein produced by strain BTA 1686.                    __________________________________________________________________________     1                             31                                               ATG                                                                               ATT                                                                               ACG                                                                               AAT                                                                               TGC                                                                               CCG                                                                               CAG                                                                               ACA                                                                               TCT                                                                               CTG                                                                               CTC                                                                               GAT                                                                               GTC                                                                               CTC                                                                               AAG                                                                               AAG                                                                               AAA                                                                               ATG                         Met                                                                               Ile                                                                               Thr                                                                               Asn                                                                               Cys                                                                               Pro                                                                               Gln                                                                               Thr                                                                               Ser                                                                               Leu                                                                               Leu                                                                               Asp                                                                               Val                                                                               Leu                                                                               Lys                                                                               Lys                                                                               Lys                                                                               Met                               61                            91                                         CGC                                                                               CAA                                                                               GCC                                                                               CGC                                                                               GAA                                                                               GAG                                                                               GCT                                                                               GAA                                                                               GCT                                                                               GCA                                                                               AAG                                                                               GAC                                                                               GAA                                                                               GCC                                                                               GAT                                                                               GAA                                                                               GTG                                                                               CAA                         Arg                                                                               Gln                                                                               Ala                                                                               Arg                                                                               Glu                                                                               Glu                                                                               Ala                                                                               Glu                                                                               Ala                                                                               Ala                                                                               Lys                                                                               Asp                                                                               Glu                                                                               Ala                                                                               Asp                                                                               Glu                                                                               Val                                                                               Gln                                     121                           151                                  CGA                                                                               CAG                                                                               CTC                                                                               GAA                                                                               GAG                                                                               GAA                                                                               CGT                                                                               AAA                                                                               AAG                                                                               CGT                                                                               GAG                                                                               GAC                                                                               GCT                                                                               GAA                                                                               GCT                                                                               GAA                                                                               GTG                                                                               GCA                         Arg                                                                               Gln                                                                               Leu                                                                               Glu                                                                               Glu                                                                               Glu                                                                               Arg                                                                               Lys                                                                               Lys                                                                               Arg                                                                               Glu                                                                               Asp                                                                               Ala                                                                               Glu                                                                               Ala                                                                               Glu                                                                               Val                                                                               Ala                                           181                           211                            GCA                                                                               TTG                                                                               AAT                                                                               CGC                                                                               CGC                                                                               ATT                                                                               GTA                                                                               TTG                                                                               GTT                                                                               GAG                                                                               GAG                                                                               GAT                                                                               TTG                                                                               GAA                                                                               CGT                                                                               ACT                                                                               GAA                                                                               GAT                         Ala                                                                               Leu                                                                               Asn                                                                               Arg                                                                               Arg                                                                               Ile                                                                               Val                                                                               Leu                                                                               Val                                                                               Glu                                                                               Glu                                                                               Asp                                                                               Leu                                                                               Glu                                                                               Arg                                                                               Thr                                                                               Glu                                                                               Asp                                                 241                                                    CGT                                                                               CTG                                                                               AAG                                                                               ATT                                                                               GCC                                                                               ACA                                                                               TCC                                                                               AAA                                                                               TTG                                                                               GAA                                                                               GAG                                                                               GCT                                                                               TCG                                                                               AAG                                                                               GCA                                                                               GCC                                                                               GAT                                                                               GAG                         Arg                                                                               Leu                                                                               Lys                                                                               Ile                                                                               Ala                                                                               Thr                                                                               Ser                                                                               Lys                                                                               Leu                                                                               Glu                                                                               Glu                                                                               Ala                                                                               Ser                                                                               Lys                                                                               Ala                                                                               Ala                                                                               Asp                                                                               Glu                         271                           301                                              GCT                                                                               GAA                                                                               CGA                                                                               GCT                                                                               CGA                                                                               AGA                                                                               TCG                                                                               TTG                                                                               GAA                                                                               AAT                                                                               CGT                                                                               GTC                                                                               GAT                                                                               GTC                                                                               GAT                                                                               GAG                                                                               GAT                                                                               CGT                         Ala                                                                               Glu                                                                               Arg                                                                               Ala                                                                               Arg                                                                               Arg                                                                               Ser                                                                               Leu                                                                               Glu                                                                               Asn                                                                               Arg                                                                               Val                                                                               Asp                                                                               Val                                                                               Asp                                                                               Glu                                                                               Asp                                                                               Arg                               331                           361                                        TGT                                                                               GCC                                                                               GAG                                                                               CTC                                                                               GAA                                                                               ACG                                                                               AAA                                                                               CTA                                                                               CGT                                                                               GAA                                                                               GCT                                                                               CAA                                                                               GCT                                                                               CTT                                                                               CTG                                                                               CAT                                                                               GAA                                                                               ACA                         Cys                                                                               Ala                                                                               Glu                                                                               Leu                                                                               Glu                                                                               Thr                                                                               Lys                                                                               Leu                                                                               Arg                                                                               Glu                                                                               Ala                                                                               Gln                                                                               Ala                                                                               Leu                                                                               Leu                                                                               His                                                                               Glu                                                                               Thr                                     391                           421                                  GAG                                                                               AGT                                                                               AAG                                                                               AGC                                                                               GAA                                                                               GAG                                                                               GTC                                                                               GCC                                                                               CGT                                                                               AAG                                                                               CTG                                                                               GCT                                                                               ATG                                                                               GTT                                                                               GAA                                                                               GCT                                                                               GAT                                                                               CTC                         Glu                                                                               Ser                                                                               Lys                                                                               Ser                                                                               Glu                                                                               Glu                                                                               Val                                                                               Ala                                                                               Arg                                                                               Lys                                                                               Leu                                                                               Ala                                                                               Met                                                                               Val                                                                               Glu                                                                               Ala                                                                               Asp                                                                               Leu                                           451                           481                            GAA                                                                               AGA                                                                               GCC                                                                               GAA                                                                               GAA                                                                               CGT                                                                               GCT                                                                               GAA                                                                               GCC                                                                               GGA                                                                               GAG                                                                               AAC                                                                               AAG                                                                               ATC                                                                               GTC                                                                               GAG                                                                               TTG                                                                               GAA                         Glu                                                                               Arg                                                                               Ala                                                                               Glu                                                                               Glu                                                                               Arg                                                                               Ala                                                                               Glu                                                                               Ala                                                                               Gly                                                                               Glu                                                                               Asn                                                                               Lys                                                                               Ile                                                                               Val                                                                               Glu                                                                               Leu                                                                               Glu                                                 511                                                    GAG                                                                               GAA                                                                               CTT                                                                               CGT                                                                               GTC                                                                               GTC                                                                               GGA                                                                               AAC                                                                               AAC                                                                               TTG                                                                               AAG                                                                               TCA                                                                               CTT                                                                               GAG                                                                               GTG                                                                               TCT                                                                               GAA                                                                               GAA                         Glu                                                                               Glu                                                                               Leu                                                                               Arg                                                                               Val                                                                               Val                                                                               Gly                                                                               Asn                                                                               Asn                                                                               Leu                                                                               Lys                                                                               Ser                                                                               Leu                                                                               Glu                                                                               Val                                                                               Ser                                                                               Glu                                                                               Glu                         541                           571                                              AAG                                                                               GCC                                                                               CTT                                                                               CAA                                                                               CGT                                                                               GAA                                                                               GAC                                                                               TCA                                                                               TAC                                                                               GAG                                                                               GAA                                                                               CAG                                                                               ATT                                                                               CGT                                                                               ACT                                                                               ATC                                                                               TCA                                                                               GCT                         Lys                                                                               Ala                                                                               Leu                                                                               Gln                                                                               Arg                                                                               Glu                                                                               Asp                                                                               Ser                                                                               Tyr                                                                               Glu                                                                               Glu                                                                               Gln                                                                               Ile                                                                               Arg                                                                               Thr                                                                               Ile                                                                               Ser                                                                               Ala                               601                           631                                        CGT                                                                               CTG                                                                               AAG                                                                               GAG                                                                               GCG                                                                               GAA                                                                               ACC                                                                               CGT                                                                               GCC                                                                               GAA                                                                               TTC                                                                               GCT                                                                               GAG                                                                               CGT                                                                               TCC                                                                               GTG                                                                               CAG                                                                               AAA                         Arg                                                                               Leu                                                                               Lys                                                                               Glu                                                                               Ala                                                                               Glu                                                                               Thr                                                                               Arg                                                                               Ala                                                                               Glu                                                                               Phe                                                                               Ala                                                                               Glu                                                                               Arg                                                                               Ser                                                                               Val                                                                               Gln                                                                               Lys                                     661                           691                                  CTC                                                                               CAG                                                                               AAG                                                                               GAA                                                                               GTC                                                                               GAC                                                                               AGA                                                                               CTG                                                                               GAG                                                                               GAT                                                                               GAA                                                                               TTG                                                                               GTA                                                                               CAT                                                                               GAG                                                                               AAG                                                                               GAG                                                                               AGA                         Leu                                                                               Gln                                                                               Lys                                                                               Glu                                                                               Val                                                                               Asp                                                                               Arg                                                                               Leu                                                                               Glu                                                                               Asp                                                                               Glu                                                                               Leu                                                                               Val                                                                               His                                                                               Glu                                                                               Lys                                                                               Glu                                                                               Arg                                           721                           751                            TAC                                                                               AAG                                                                               GCA                                                                               ATT                                                                               TCC                                                                               GAG                                                                               GAG                                                                               CTT                                                                               GAC                                                                               TCG                                                                               ACC                                                                               TTC                                                                               CAA                                                                               GAA                                                                               CTG                                                                               TCC                                                                               GGC                                                                               TAT                         Tyr                                                                               Lys                                                                               Ala                                                                               Ile                                                                               Ser                                                                               Glu                                                                               Glu                                                                               Leu                                                                               Asp                                                                               Ser                                                                               Thr                                                                               Phe                                                                               Gln                                                                               Glu                                                                               Leu                                                                               Ser                                                                               Gly                                                                               Tyr                         __________________________________________________________________________

EXAMPLE 6 Expression of H. contortus Antigen by Recombinant Organisms

Recombinant E. coli strains BTA 1637, BTA 1638, BTA 1684 and BTA 1686 were grown in TSB containing ampicillin (50 μ/ml) until an OD of approximately 1. Iscoropylthiogalactopyranoside (IPTG) was then added to a final concentration of 10 mM and the incubation was continued for a further 8-16 h. The cells were collected by centrifugation, lysed by sonication and the sonicate was centrifuged at 13,000×g for 10 min. The pellet and the supernatants were subjected to SDS-PAGE analysis (Laemmli 1970) and immunoblotting using antisera raised in rabbits against the T. colubriformis 41 kD antigen. Immune complexes were detected using ¹²⁵ I-protein A or goat-anti-rabbit antibodies conjugated to alkaline phosphatase. The fusion proteins were found mainly in the pellets of the cell lysates in the form of inclusion bodies. BTA 1637 produced a fusion of approximately 120 kj which was detected on both Coomassie stained gels and the protein blots. BTA 1638 produced a fusion protein of approximately 148 kd which could be detected on a protein blot but not readily on a Coomassie-blue stained gel. BTA 1684 and BTA 1686 produced proteins of approximately 44 kd and 30 kd respectively which were readily visualized on both stained gels and protein blots. The molecular weight of each product was approximately that predicted from analysis of the DNA sequence in all cases.

To produce material for animal trials, BTA 1684 was inoculated into a fermenter containing minimal media and auxotrophic requirements and fermented until the OD reached 15.5. IPTG was added to a final concentration of 1 mM, a tryptone yeast extract enriched media was added and a glucose feed begun. 154 hours post inoculation the cells were collected by centrifugation, resuspended and lysed by four passages through a french presse at 9,000 psi. The lysate was centrifuged at approximately and the pellet was resuspended in 8M urea, 100 mM DTT, 20 mM tris-HCI ph8.0. Following centrifugation, the supernatant was passed over a DEAE sepharase column which was resolved with a 0-1M gradient of NaCl. Fractions containing the fusion protein were pooled and desalted on a Sephacryl S-400 HR column in 1M acetic acid. The fractions containing the fusion protein were again pooled, bound to a VydaK C₄ column in 0.1% Trifluoroacetic acid (TFA) which was resolved with a linear gradient of 30%-45% acetonitrile. Fractions containing the fusion protein were pooled and exchanged into 1M acetic acid for storage. An aliquot of the purified material was taken and the N-terminal amino acid sequence determined using an Applied Biosystems amino acid anlayser. The first 20 amino acids were as predicted vis Met Ile Thr Asn X Pro Gly Asp Leu Glu Phe X Phe Gly Ala Ala Thr Asp Glu Ser. The amino acid analysis and SDS PAGE analysis would indicate the recombinant product to be at least 85% pure.

EXAMPLE 7 Vaccination/Challenge Trials

A group of 10 sheep reared worm free were vaccinated with the recombinant antigen purified from strain BTA 1684 described in Example 6. Each animal received two vaccinations subcutaneously. The first vaccination contained approximately 1 mg of protein emulsified in Freunds complete adjuvant and the second 28 days later contained approximately 250 μg emulsified in Freunds incomplete adjuvant. Two additional groups of ten animals were used, the first as infection controls received no vaccinations and the second as adjuvant controls received adjuvant alone. Sixty three days after the first vaccination, all animals were infected per os with 15,000 infective L3 larvae of H. contortus. Faecal samples were taken on days 22, 28, 36, 42, 50, 56 and 63 post challenge and eggs per g. faeces were determined. In addition, haematocrit values were assayed on days 37, 56 and 63 post infection. All animals were slaughtered on day 63 and the number of adult H. contortus in the abomasum was counted. The results (Tables 9, 10 and 11) show that vaccination with the recombinant antigen had given significant protection to the sheep against parasitism.

Throughout the period of the experiment the vaccinated group had a reduced egg count compared to the control groups: approximately 25% over the first 4 time points and approximately 60% over the last 3 time points, average overall of 40% reduced egg counts. At all three time points where the haematocrit values were measured the vaccinated group were higher and therefore the animals were less distressed. At slaughter the vaccinated group containing fewer worms, on average 52% lower than the control groups.

This experiment confirms that an immune response raised against the recombinant 41 kD protein was capable of reducing egg production and worm burden of infected sheep by greater than 50% and this correlated with reduced blood loss in these animals.

NOTE: Two animals had to be removed from the trial for ethical reasons when their haematocrit values dropped below an arbitrarily chosen value of 15. One of these animals came from the challenge control group and was slaughtered on day 52 (faecal egg count (day 50) 168,000 worm burden 6440). The second, from the vaccinate group, was slaughtered on day 34 (faecal egg count (day 28) 62,400, worm burden 6755). The data for these animals has not been included in Tables 9-11. As can be seen from analysis of the data in Table 9, the major effect of vaccination was that of an accelerated expulsion of the parasites. The effects of vaccination on days 22-42 were very minor on the whole group so it is not surprising that some animals were poorly protected during this early period of time. It is to be understood that as the vaccination regime is optimised, the degree of protection obtained would be expected to increase to such an extent that a highly effective vaccine based on this antigen could be developed.

                  TABLE 9                                                          ______________________________________                                         FAECAL EGG COUNTS                                                              Days                                                                           Post Challenge   Adjuvant              Average                                 Chal-                                                                               Controls    Controls    Vaccinates                                                                               % Pro-                                  lenge                                                                               (n = 9)     (n = 10)    (n = 9)   tection                                 ______________________________________                                         22   3,311 ± 2,541                                                                           1,505 ± 2,019                                                                           1,611 ± 1,389                                                                         33%                                     28   15,078 ± 10,566                                                                         17,740 ± 10,559                                                                         13,311 ± 8,704                                                                        20%                                     34   50,467 ± 28,004                                                                         54,230 ± 30,102                                                                         45,400 ± 32,702                                                                       13%                                     42   32,906 ± 19,439                                                                         27,405 ± 18,028                                                                         22,094 ± 14,003                                                                       27%                                     50   40,289 ± 31,353                                                                         30,620 ± 19,911                                                                         14,439 ± 12,436                                                                       59%                                     56   19,894 ± 20,383                                                                         23,535 ± 16,969                                                                         7,850 ± 9,614                                                                         64%                                     63   29,417 ± 23,922                                                                         41,385 ± 50,019                                                                         13,817 ± 13,888                                                                       61%                                                                            =40%                                    ______________________________________                                    

                  TABLE 10                                                         ______________________________________                                         PACKED CELL VOLUMES                                                                      CHALLENGE   ADJUVANT                                                 DAYS POST CONTROLS    CONTROLS   VACCINATES                                    CHALLENGE (n = 9)     (n = 10)   (n = 9)                                       ______________________________________                                         37        22.4 ± 4.3                                                                              22.8 ± 4.6                                                                             24.2 ± 3.6                                 56        22.0 ± 4.3                                                                              24.9 ± 5.2                                                                             26.7 ± 3.0                                 63        24.6 ± 4.8                                                                              25.3 ± 4.8                                                                             27.6 ± 3.9                                 ______________________________________                                    

                  TABLE 11                                                         ______________________________________                                         WORM COUNTS AT SLAUGHTER                                                       CHALLENGE                                                                               ADJUVANT             AVERAGE T. Test                                  CONTROLS CONTROLS  VACCINATES % PRO-  SIGNIF-                                  (n = 9)  (n = 10)  (n = 9)    TECTION ICANCE                                   ______________________________________                                         3700 ± 2243                                                                          3776 +3196                                                                               1804 ± 1146                                                                            52%     <0.20                                    ______________________________________                                    

EXAMPLE 8 Cross Species Protection by 41 Kd Cloned Antigen

2 groups of 5 guinea pigs were vaccinated with the recombinant antigen purified from strain BTA 1684 as described in Example 6. Each group received on vaccination intraperitoneally. One group received 100 μg of protein in buffer and the second group received 100 μg of protein emulsified in incomplete Freunds adjuvant. One additional group of 4 animals, infection controls received no vaccination. 28 days after the vaccination, all animals were infected per os with 2000 infective L₃ larvae of T. colubriformis. 14 days later, all animals were slaughtered and the number of worms were counted. The results (Table 12) show that vaccination with the antigen alone gave 31% protection while vaccination with the antigen in incomplete Freunds adjuvant gave 46% protection.

These figures are similar to the protection provided by the native 41 kD antigens isolated from T. colubriformis (Example 4).

                  TABLE 12                                                         ______________________________________                                                                        T-test                                                       Worm count                                                                             Protection                                                                               Significance                                    ______________________________________                                         Controls  (n = 4)  1237 ± 142                                               44 kD alone                                                                              (n = 5)  853 ± 302                                                                             31%     <0.050                                    44 kD in IFA                                                                             (n = 5)  667 ± 195                                                                             46%     <0.005                                    ______________________________________                                    

These results demonstrate that the H. contortus 41 kD antigen is capable of giving significant cross species protection to animals against infection with T. colubriformis. By extension it is likely that similar cross species protection could be afforded by vaccination with the recombinant antigens described herein or by vaccination with recombinant antigens derived from the other species of parasitic nematodes using the technology described in this work.

It is anticipated that the degree of protection afforded to the host animal would be increased substantially to provide an effective vaccine following optimization of the antigen purification, formulation and vaccination procedures.

INDUSTRIAL APPLICATIONS

The current invention provides a description of a protein and the DNA sequence coding for that protein which can be used as an effective vaccine for protection against parasitism of animals by Trichostrongylus colubriformis and Haemonchus contortus.

Furthermore it has been demonstrated that antibodies raised against the purified protein isolated from Trichostrongylus colubriformis and the DNA sequences coding for this protein can be used to identify the related polypeptide and gene coding for that polypeptide from species of parasitic nematode other than Trichostrongylus colubriformis. Thus it is claimed that the same DNA sequence and antibodies can be used to identify related proteins and genes coding for those proteins in a range of other species of nematode which are parasitic to man and domestic animals and that these proteins will provide effective vaccines against parasitism by those species of nematode whether isolated from the parasite itself or produced by recombinant DNA technology. Species of parasites and hosts they may infect include for example.

Trichinella spiralis or Ancylostoma canium infections of man, Strongylus vulgaris infections of horses, Trichostrongylus colubriformis infections of sheep, Haemonchus contortus infects of goats, Ostertagia ostertagi infections of cattle, Ascaris suum or Trichinella spiralis infections of pigs, Toxascaris leonina or Uncinaria stenocephala infections of cats and Ancylostoma canium or Trichuris vulpis infections of dogs as well as infections of the circulatory system of man by larvae of Taxocara spp. infection by Necator americanus, Ancylostoma duodenale, Ascaris lumbricoides, Trichuris trichiura, Enterobius vermicularus, Strongyloides stercorals or Wucheria bancrofti and of the circulatory system of dogs by Dirofilaria immitis as well as infections of the circulatory system, urogenital system, respiratory system, skin and subcutaneous tissues of these and other species of animal. It should be noted that this list is by no means complete.

REFERENCES

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Benton, W D and R W, Davis, 1977, Science, 196, 180-182.

Dineen et al 1977 Int. J. Parasitol, 7, 211-215.

Dineen J K and Wagland B M (1982)--Immunoregulation of parasites in natural host-parasite systems--with special reference to the gastrointestinal nematodes of sheep. In "Biology and Control of Endoparasites" (Eds. L E A Symons, A D Donald and J K Dineen), Academic Press, pp 297-323.

Dineen J K (1984)--Immunological control of helminthiasis by genetic manipulation of host and parasite. In "Immunogenetic approaches to the control of endoparasites" (Eds J K Dineen and P M Outteridge), CSIRO, Australia, pp 2-8.

Feinberg, A P and Vogelstein, B 1984, Anal. Biochem. 137, 266-267.

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Howe J G and Hershey J W B (1981)--A sensitive immunoblotting method for measuring protein synthesis initiation factor levels in lysates of Escherichia coli--J of Biological Chemistry 256, 12836-12839.

Huynh, T V, R A, Young and R W, Davis, pp 49-78 in DNA cloning Vol. 1 1985 ed D M, Glover.

Johnson et al, 1984, Gene. Anal. Tech. 1, 3-8.

Kohler P (1986)--Progress in molecular parasitology--Experientia 42, 377-386.

Laemmli, U K, 1970, Nature, 227, 680-685.

Laemmli U K (1970)--Cleavage of structural proteins during the assembly of the head of bacteriophage T4--Nature (London) 227, 680-685.

McKern N M, O'Donnell I J, Stewart D J and Clark B L (1985)--Primary structure of pilin protein from Bacteroides nodosus strain 216: comparison with corresponding protein from strain 198. J General Microbiology 131, 1-6.

Maniatis T, Fritsch E F and Sambrook J (1982) Molecular Cloning: A Laboratory Manual (Cold Spring Harbour Laboratory).

Maruyama, T, T Gojobori, S-I, Aota and T Idemura, 1986, Nucleic Acids Research, 14, Supplement r151-r197.

May R B (1985)--Evolution of pesticide resistance--Nature (London) 315, 12-13.

Munn, E and Greenwood, B 1987, Parasitology.

Neilson, J T M, 1975, Int. J. Parasitol. 5, 427-430.

Neilson and Van de Walle, 1987 Vet. Parasitol. 211-211.

O'Donnell I J (1978)--A search for a simple Karatin--Fractionation and peptide mapping of proteins from feature keratins. Aust. J. Biol. Sci. 26, 401-13.

O'Donnell I J, Dineen J K, Rothwell T L W and Marshall R C (1985)--Attempts to probe the antigens and protective immunogens of Trichostrongylus colubriformis in immunoblots with sera from infected and hyperimmune sheep and high- and low-responder guinea pigs--International Journal for Parasitology 15, 129-136.

Payne J W (1973)--Polymerization of proteins with glutaraldehyde--Biochem. J 135, 67-873.

Racusen D (1979)--Glycoprotein detection in polyacrylamide gel with thyml and sulphuric acid--Analytical Biochemistry 99, 474-476.

Reed, K C, BIO-RAD Bulletin 1232.

Rothwell T L W (1978)--Vaccination against the nematode Trichostrongylus colubriformis--III. Some observations on factors influencing immunity to infection in vaccinated guinea-pigs--International Journal for Parasitology 8, 33-37.

Rothwell T L W and Griffiths D A (1977)--Comparison of the kinetics of expulsion of Trichostrongylus colubriformis from previously uninfected, reinfected, and vaccinated guinea pigs, J of Parasitology 63, 761-762.

Rothwell T L W and Love R L (1974)--Vaccination against the nematode Trichostrongylus colubriformis--I Vaccination of guinea-pigs with worm homogenates and soluble products released during in vitro maintenance--International Journal for Parasitology 4, 293-299.

Rubin R W and Leonardi C L (1983)--Two-dimensional polyacrylamide gel electrophororesis of membrane proteins. In `Methods in Enzymology` (Eds Sidney and Betta Feischer) 96, 184-192.

Ruther U & Muller-Hill B (1983) EMBO J 1,1217.

Sanger, F, Coulson, A R, Barrell, B G, Smith, A J H and Roe, B A 1980, J. Mol. Biol. 143, 161-178.

Silverstein D S and Despommier D D (1985)--Effect on Trichinella spiralis of host responses to purified antigens--Science 227, 948-950.

Stearne P A, van Driel I R, Grego B, Simpson R J and Goding J W (1985)--The murine plasma cell antigen PC-1: Purification and partial amino acid sequence. J of Immunology 134, 443-448.

Towoin H, Staenelin T and Gordon J (1979) PNAS 76(9), 4350-4354.

Wallace, R B, P F, Johnson, S Taneka, M Schold, K Itakura and J Abelson, 1985 Science 209, 1396-1400.

Yanisch-Perron, C., Vierira, J. and Messing, J. (1985) Gene 33, 103-119. 

We claim:
 1. An isolated parasitic nematode tropomyosin protein capable of conferring protective immunity against infections of a host by a parasitic nematode species, wherein said tropomyosin protein binds to antibodies raised against a protein isolated from Trichostrongylus colubriformis,wherein said Trichostrongylus colubriformis protein has an approximate molecular weight of 41 kD as determined by SDS-PAGE, and comprises the amino acid sequence: ##STR14## .
 2. A protein according to claim 1, wherein said nematode tropomyosin protein is encoded by a DNA molecule having the sequence;

    __________________________________________________________________________        ATG                                                                               GAC                                                                               GCC                                                                               ATC                                                                               AAG                                                                               AAG                                                                               AAG                                                                               ATG                                                                               CAG                                                                               GCG                                                                               ATG                                                                               AAG                                                                               ATC                                                                               GAG                                                                               AAG                               GAC                                                                               AAT                                                                               GCT                                                                               CTC                                                                               GAT                                                                               CGA                                                                               GCC                                                                               GAT                                                                               GCC                                                                               GCC                                                                               GAA                                                                               GAG                                                                               AAA                                                                               GTC                                                                               CGT                                                                               CAA                               ATT                                                                               ACC                                                                               GAA                                                                               AAG                                                                               TTG                                                                               GAG                                                                               CGA                                                                               GTT                                                                               GAA                                                                               GAA                                                                               GAG                                                                               CTC                                                                               CGT                                                                               GAC                                                                               ACA                                                                               CAA                               AAG                                                                               AAA                                                                               ATG                                                                               ATG                                                                               CAA                                                                               ACA                                                                               GAA                                                                               AAC                                                                               GAT                                                                               TTG                                                                               GAC                                                                               AAG                                                                               GCT                                                                               CAG                                                                               GAA                                                                               GAT                               TTG                                                                               GCT                                                                               GCA                                                                               GCC                                                                               ACC                                                                               AGC                                                                               CAG                                                                               TTG                                                                               GAA                                                                               GAG                                                                               AAA                                                                               GAG                                                                               AAG                                                                               AAA                                                                               GTG                                                                               CAA                               GAG                                                                               GCT                                                                               GAG                                                                               GCA                                                                               GAG                                                                               GTA                                                                               GCT                                                                               GCC                                                                               CTG                                                                               AAC                                                                               CGT                                                                               CGC                                                                               ATG                                                                               ACT                                                                               CTT                                                                               CTC                               GAA                                                                               GAA                                                                               GAG                                                                               CTT                                                                               GAA                                                                               CGT                                                                               GCT                                                                               GAA                                                                               CAA                                                                               CGT                                                                               TTG                                                                               AAG                                                                               ATC                                                                               GCC                                                                               ACT                                                                               GAA                               AAA                                                                               CTC                                                                               GAA                                                                               GAG                                                                               GCC                                                                               ACT                                                                               CAC                                                                               AAT                                                                               GTC                                                                               GAC                                                                               GAG                                                                               TCC                                                                               GAG                                                                               CGT                                                                               GTA                                                                               CGC                               AAA                                                                               GTG                                                                               ATG                                                                               GAG                                                                               AAC                                                                               GGC                                                                               TCA                                                                               TTC                                                                               CAA                                                                               GAT                                                                               GAG                                                                               GAG                                                                               CGT                                                                               GCC                                                                               AAC                                                                               ACG                               ATT                                                                               GAA                                                                               GCT                                                                               CAA                                                                               CTG                                                                               AAG                                                                               GAA                                                                               GCC                                                                               CAG                                                                               ATG                                                                               CTT                                                                               GCA                                                                               GAG                                                                               GAA                                                                               GCC                                                                               GAC                               CGA                                                                               AAA                                                                               TAC                                                                               GAT                                                                               GAG                                                                               GTC                                                                               GCC                                                                               CGT                                                                               AAA                                                                               TTG                                                                               GCC                                                                               ATG                                                                               GTT                                                                               GAA                                                                               GCT                                                                               GAT                               CTT                                                                               GAA                                                                               AGG                                                                               GCC                                                                               GAA                                                                               GAA                                                                               CGT                                                                               GCT                                                                               GAA                                                                               GCC                                                                               GGA                                                                               GAG                                                                               AAC                                                                               AAG                                                                               ATC                                                                               GTC                               GAA                                                                               TTG                                                                               GAA                                                                               GAG                                                                               GAA                                                                               TTG                                                                               CGT                                                                               GTC                                                                               GTC                                                                               GGA                                                                               AAT                                                                               AAC                                                                               CTG                                                                               AAA                                                                               TCA                                                                               CTC                               GAA                                                                               GTG                                                                               TCC                                                                               CAA                                                                               GAA                                                                               AAG                                                                               GCA                                                                               CTG                                                                               CAA                                                                               CGT                                                                               GAG                                                                               GAC                                                                               TCA                                                                               TAC                                                                               GAA                                                                               GAG                               CAG                                                                               ATT                                                                               CGT                                                                               ACC                                                                               ATT                                                                               TCA                                                                               TCT                                                                               CGT                                                                               CTG                                                                               AAG                                                                               GAG                                                                               GCG                                                                               GAA                                                                               ACC                                                                               CGT                                                                               GCT                               GAA                                                                               TTC                                                                               GCC                                                                               GAG                                                                               CGC                                                                               TCC                                                                               GTG                                                                               CAG                                                                               AAG                                                                               CTC                                                                               CAG                                                                               AAG                                                                               GAA                                                                               GTC                                                                               GAC                                                                               AGA                               CTC                                                                               GAG                                                                               GAT                                                                               GAA                                                                               TTG                                                                               GTA                                                                               CAT                                                                               GAG                                                                               AAG                                                                               GAG                                                                               AGA                                                                               TAC                                                                               AAG                                                                               GCG                                                                               ATT                                                                               TCC                               GAG                                                                               GAG                                                                               CTT                                                                               GAC                                                                               TCG                                                                               ACC                                                                               TTC                                                                               CAA                                                                               GAA                                                                               CTC                                                                               TCC                                                                               GGC                                                                               TAT                                        __________________________________________________________________________


3. A nematode tropomyosin protein according to claim 1 wherein said parasitic nematode species is selected from the group consisting of Trichinella spiralis, Ancylostoma caninum, Strongylus vulgaris, Ostertagia ostertagi, Ascaris suum, Trichinella spiralis, Toxascaris leonia, Uncinaria stenocephalia, Ancylostoma canium, Trichuris vulpis, Dirofilaria immitis, a larva of Toxocara spp, Trichostrongylus colubriformis, Haemonchus contortus, Necator americanus, Ancylostoma duodenale, Ascaris lumbricoides, Trichuris trichiura, Enterobius vermicularus, Strongyloides stercorals or Wuchereria bancrofti.
 4. A protein according to claim 1 wherein said nematode tropomyosin protein is derived from T. colubriformis or H. contortus.
 5. A protein according to claim 1, wherein said nematode tropomyosin protein comprises the sequence:

    __________________________________________________________________________        Lys                                                                               Lys                                                                               Lys                                                                               Met                                                                               Gln                                                                               Ala                                                                               Met                                                                               Lys                                                                               Ile                                                                               Glu                                                                               Lys                                                                               Asp                                                                               Asn                                                                               Ala                                                                               Leu                               Asp                                                                               Arg                                                                               Ala                                                                               Asp                                                                               Ala                                                                               Ala                                                                               Glu                                                                               Glu                                                                               Lys                                                                               Val                                                                               Arg                                                                               Gln                                                                               Ile                                                                               Tru                                                                               Glu                                                                               Lys                               Leu                                                                               Glu                                                                               Arg                                                                               Val                                                                               Glu                                                                               Glu                                                                               Glu                                                                               Leu                                                                               Arg                                                                               Asp                                                                               Thr                                                                               Gln                                                                               Lys                                                                               Lys                                                                               Met                                                                               Met                               Gln                                                                               Thr                                                                               Glu                                                                               Asn                                                                               Asp                                                                               Leu                                                                               Asp                                                                               Lys                                                                               Ala                                                                               Gln                                                                               Glu                                                                               Asp                                                                               Leu                                                                               Ala                                                                               Ala                                                                               Ala                               Thr                                                                               Ser                                                                               Gln                                                                               Leu                                                                               Glu                                                                               Glu                                                                               Lys                                                                               Glu                                                                               Lys                                                                               Lys                                                                               Val                                                                               Gln                                                                               Glu                                                                               Ala                                                                               Glu                                                                               Ala                               Glu                                                                               Val                                                                               Ala                                                                               Ala                                                                               Leu                                                                               Asn                                                                               Arg                                                                               Arg                                                                               Met                                                                               Thr                                                                               Leu                                                                               Leu                                                                               Glu                                                                               Glu                                                                               Glu                                                                               Leu                               Glu                                                                               Arg                                                                               Ala                                                                               Glu                                                                               Glu                                                                               Arg                                                                               Leu                                                                               Lys                                                                               Ile                                                                               Ala                                                                               Tru                                                                               Glu                                                                               Lys                                                                               Leu                                                                               Glu                                                                               Glu                               Ala                                                                               Thr                                                                               His                                                                               Asn                                                                               Val                                                                               Asp                                                                               Glu                                                                               Ser                                                                               Glu                                                                               Arg                                                                               Val                                                                               Arg                                                                               Lys                                                                               Val                                                                               Met                                                                               Glu                               Asn                                                                               Gly                                                                               Ser                                                                               Phe                                                                               Gln                                                                               Asp                                                                               Glu                                                                               Glu                                                                               Arg                                                                               Ala                                                                               Asn                                                                               Thr                                                                               Ile                                                                               Glu                                                                               Ala                                                                               Gln                               Leu                                                                               Lys                                                                               Glu                                                                               Ala                                                                               Gln                                                                               Met                                                                               Leu                                                                               Ala                                                                               Glu                                                                               Glu                                                                               Ala                                                                               Asp                                                                               Arg                                                                               Lys                                                                               Tyr                                                                               Asp                               Glu                                                                               Val                                                                               Ala                                                                               Arg                                                                               Lys                                                                               Leu                                                                               Ala                                                                               Met                                                                               Val                                                                               Glu                                                                               Ala                                                                               Asp                                                                               Leu                                                                               Glu                                                                               Arg                                                                               Ala                               Glu                                                                               Glu                                                                               Arg                                                                               Ala                                                                               Glu                                                                               Ala                                                                               Gly                                                                               Glu                                                                               Asn                                                                               Lys                                                                               Ile                                                                               Val                                                                               Glu                                                                               Leu                                                                               Glu                                                                               Glu                               Glu                                                                               Leu                                                                               Arg                                                                               Val                                                                               Val                                                                               Gly                                                                               Asn                                                                               Asn                                                                               Leu                                                                               Lys                                                                               Ser                                                                               Leu                                                                               Glu                                                                               Val                                                                               Ser                                                                               Glu                               Glu                                                                               Lys                                                                               Ala                                                                               Leu                                                                               Gln                                                                               Arg                                                                               Glu                                                                               Asp                                                                               Ser                                                                               Tyr                                                                               Glu                                                                               Glu                                                                               Gln                                                                               Ile                                                                               Arg                                                                               Thr                               Ile                                                                               Ser                                                                               Ser                                                                               Arg                                                                               Leu                                                                               Lys                                                                               Glu                                                                               Ala                                                                               Glu                                                                               Thr                                                                               Arg                                                                               Ala.                                          __________________________________________________________________________


6. A protein according to claim 1, wherein said nematode tropomyosin protein comprises the sequence:

    __________________________________________________________________________        Gln                                                                               Thr                                                                               Ser                                                                               Leu                                                                               Leu                                                                               Asp                                                                               Val                                                                               Leu                                                                               Lys                                                                               Lys                                                                               Lys                                                                               Met                                                                               Arg                                                                               Gln                                                                               Ala                               Arg                                                                               Glu                                                                               Glu                                                                               Ala                                                                               Glu                                                                               Ala                                                                               Ala                                                                               Lys                                                                               Asp                                                                               Glu                                                                               Ala                                                                               Asp                                                                               Glu                                                                               Val                                                                               Gln                                                                               Arg                               Gln                                                                               Leu                                                                               Glu                                                                               Glu                                                                               Glu                                                                               Arg                                                                               Lys                                                                               Lys                                                                               Arg                                                                               Glu                                                                               Asp                                                                               Ala                                                                               Glu                                                                               Ala                                                                               Glu                                                                               Val                               Ala                                                                               Ala                                                                               Leu                                                                               Asn                                                                               Arg                                                                               Arg                                                                               Ile                                                                               Val                                                                               Leu                                                                               Val                                                                               Glu                                                                               Glu                                                                               Asp                                                                               Leu                                                                               Glu                                                                               Arg                               Thr                                                                               Glu                                                                               Asp                                                                               Arg                                                                               Leu                                                                               Lys                                                                               Ile                                                                               Ala                                                                               Thr                                                                               Ser                                                                               Lys                                                                               Leu                                                                               Glu                                                                               Glu                                                                               Ala                                                                               Ser                               Lys                                                                               Ala                                                                               Ala                                                                               Asp                                                                               Glu                                                                               Ala                                                                               Glu                                                                               Arg                                                                               Ala                                                                               Arg                                                                               Arg                                                                               Ser                                                                               Leu                                                                               Glu                                                                               Asn                                                                               Arg                               Val                                                                               Asp                                                                               Val                                                                               Asp                                                                               Glu                                                                               Asp                                                                               Arg                                                                               Cys                                                                               Ala                                                                               Glu                                                                               Leu                                                                               Glu                                                                               Thr                                                                               Lys                                                                               Leu                                                                               Arg                               Glu                                                                               Ala                                                                               Gln                                                                               Ala                                                                               Leu                                                                               Leu                                                                               His                                                                               Glu                                                                               Thr                                                                               Glu                                                                               Ser                                                                               Lys                                                                               Ser                                                                               Glu                                                                               Glu                                                                               Val                               Ala                                                                               Arg                                                                               Lys                                                                               Leu                                                                               Ala                                                                               Met                                                                               Val                                                                               Glu                                                                               Ala                                                                               Asp                                                                               Leu                                                                               Glu                                                                               Arg                                                                               Ala                                                                               Glu                                                                               Glu                               Arg                                                                               Ala                                                                               Glu                                                                               Ala                                                                               Gly                                                                               Glu                                                                               Asn                                                                               Lys                                                                               Ile                                                                               Val                                                                               Glu                                                                               Leu                                                                               Glu                                                                               Glu                                                                               Glu                                                                               Leu                               Arg                                                                               Val                                                                               Val                                                                               Gly                                                                               Asn                                                                               Asn                                                                               Leu                                                                               Lys                                                                               Ser                                                                               Leu                                                                               Glu                                                                               Val                                                                               Ser                                                                               Glu                                                                               Glu                                                                               Lys                               Ala                                                                               Leu                                                                               Gln                                                                               Arg                                                                               Glu                                                                               Asp                                                                               Ser                                                                               Tyr                                                                               Glu                                                                               Glu                                                                               Glu                                                                               Ile                                                                               Arg                                                                               Thr                                                                               Ile                                                                               Ser                               Ala                                                                               Arg                                                                               Leu                                                                               Lys                                                                               Glu                                                                               Ala                                                                               Glu                                                                               Thr                                                                               Arg                                                                               Ala                                                                               Glu                                                                               Phe                                                                               Ala                                                                               Glu                                                                               Arg                                                                               Ser                               Val                                                                               Gln                                                                               Lys                                                                               Leu                                                                               Gln                                                                               Lys                                                                               Glu                                                                               Val                                                                               Asp                                                                               Arg                                                                               Leu                                                                               Glu                                                                               Asp                                                                               Glu                                                                               Leu                                                                               Val                               His                                                                               Glu                                                                               Lys                                                                               Glu                                                                               Arg                                                                               Tyr                                                                               Lys                                                                               Ala                                                                               Ile                                                                               Ser                                                                               Glu                                                                               Glu                                                                               Leu                                                                               Asp                                                                               Ser                                                                               Thr                               Phe                                                                               Gln                                                                               Glu                                                                               Leu                                                                               Ser                                                                               Gly                                                                               Tyr.                                                         __________________________________________________________________________


7. A method for preparing a nematode tropomyosin protein capable of conferring protective immunity against infection of a host by a parasitic nematode species, said method comprising the steps of (A) culturing a host cell transformed with a DNA sequence encoding said protein, wherein said DNA sequence comprises the sequence set forth below, and (B) isolating said protein from said microorganism:

    __________________________________________________________________________        ATG                                                                               GAC                                                                               GCC                                                                               ATC                                                                               AAG                                                                               AAG                                                                               AAG                                                                               ATG                                                                               CAG                                                                               GCG                                                                               ATG                                                                               AAG                                                                               ATC                                                                               GAG                                                                               AAG                               GAC                                                                               AAT                                                                               GCT                                                                               CTC                                                                               GAT                                                                               CGA                                                                               GCC                                                                               GAT                                                                               GCC                                                                               GCC                                                                               GAA                                                                               GAG                                                                               AAA                                                                               GTC                                                                               CGT                                                                               CAA                               ATT                                                                               ACC                                                                               GAA                                                                               AAG                                                                               TTG                                                                               GAG                                                                               CGA                                                                               GTT                                                                               GAA                                                                               GAA                                                                               GAG                                                                               CTC                                                                               CGT                                                                               GAC                                                                               ACA                                                                               CAA                               AAG                                                                               AAA                                                                               ATG                                                                               ATG                                                                               CAA                                                                               ACA                                                                               GAA                                                                               AAC                                                                               GAT                                                                               TTG                                                                               GAC                                                                               AAG                                                                               GCT                                                                               CAG                                                                               GAA                                                                               GAT                               TTG                                                                               GCT                                                                               GCA                                                                               GCC                                                                               ACC                                                                               AGC                                                                               CAG                                                                               TTG                                                                               GAA                                                                               GAG                                                                               AAA                                                                               GAG                                                                               AAG                                                                               AAA                                                                               GTG                                                                               CAA                               GAG                                                                               GCT                                                                               GAG                                                                               GCA                                                                               GAG                                                                               GTA                                                                               GCT                                                                               GCC                                                                               CTG                                                                               AAC                                                                               CGT                                                                               CGC                                                                               ATG                                                                               ACT                                                                               CTT                                                                               CTC                               GAA                                                                               GAA                                                                               GAG                                                                               CTT                                                                               GAA                                                                               CGT                                                                               GCT                                                                               GAA                                                                               CAA                                                                               CGT                                                                               TTG                                                                               AAG                                                                               ATC                                                                               GCC                                                                               ACT                                                                               GAA                               AAA                                                                               CTC                                                                               GAA                                                                               GAG                                                                               GCC                                                                               ACT                                                                               CAC                                                                               AAT                                                                               GTC                                                                               GAC                                                                               GAG                                                                               TCC                                                                               GAG                                                                               CGT                                                                               GTA                                                                               CGC                               AAA                                                                               GTG                                                                               ATG                                                                               GAG                                                                               AAC                                                                               GGC                                                                               TCA                                                                               TTC                                                                               CAA                                                                               GAT                                                                               GAG                                                                               GAG                                                                               CGT                                                                               GCC                                                                               AAC                                                                               ACG                               ATT                                                                               GAA                                                                               GCT                                                                               CAA                                                                               CTG                                                                               AAG                                                                               GAA                                                                               GCC                                                                               CAG                                                                               ATG                                                                               CTT                                                                               GCA                                                                               GAG                                                                               GAA                                                                               GCC                                                                               GAC                               CGA                                                                               AAA                                                                               TAC                                                                               GAT                                                                               GAG                                                                               GTC                                                                               GCC                                                                               CGT                                                                               AAA                                                                               TTG                                                                               GCC                                                                               ATG                                                                               GTT                                                                               GAA                                                                               GCT                                                                               GAT                               CTT                                                                               GAA                                                                               AGG                                                                               GCC                                                                               GAA                                                                               GAA                                                                               CGT                                                                               GCT                                                                               GAA                                                                               GCC                                                                               GGA                                                                               GAG                                                                               AAC                                                                               AAG                                                                               ATC                                                                               GTC                               GAA                                                                               TTG                                                                               GAA                                                                               GAG                                                                               GAA                                                                               TTG                                                                               CGT                                                                               GTC                                                                               GTC                                                                               GGA                                                                               AAT                                                                               AAC                                                                               CTG                                                                               AAA                                                                               TCA                                                                               CTC                               GAA                                                                               GTG                                                                               TCC                                                                               CAA                                                                               GAA                                                                               AAG                                                                               GCA                                                                               CTG                                                                               CAA                                                                               CGT                                                                               GAG                                                                               GAC                                                                               TCA                                                                               TAC                                                                               GAA                                                                               GAG                               CAG                                                                               ATT                                                                               CGT                                                                               ACC                                                                               ATT                                                                               TCA                                                                               TCT                                                                               CGT                                                                               CTG                                                                               AAG                                                                               GAG                                                                               GCG                                                                               GAA                                                                               ACC                                                                               CGT                                                                               GCT                               GAA                                                                               TTC                                                                               GCC                                                                               GAG                                                                               CGC                                                                               TCC                                                                               GTG                                                                               CAG                                                                               AAG                                                                               CTC                                                                               CAG                                                                               AAG                                                                               GAA                                                                               GTC                                                                               GAC                                                                               AGA                               CTC                                                                               GAG                                                                               GAT                                                                               GAA                                                                               TTG                                                                               GTA                                                                               CAT                                                                               GAG                                                                               AAG                                                                               GAG                                                                               AGA                                                                               TAC                                                                               AAG                                                                               GCG                                                                               ATT                                                                               TCC                               GAG                                                                               GAG                                                                               CTT                                                                               GAC                                                                               TCG                                                                               ACC                                                                               TTC                                                                               CAA                                                                               GAA                                                                               CTC                                                                               TCC                                                                               GGC                                                                               TAT.                                       __________________________________________________________________________


8. A protein according to claim 7, wherein said tropomyosin protein is a fusion protein that also comprises a peptide sequence homologous to said host cell.
 9. A method according to claim 7, wherein said host cell is selected from the group consisting of bacteria, yeast, fungi other than yeast, vertebrate and insect cells.
 10. A method according to claim 9, wherein said host cell is a bacterial cell.
 11. A method according to claim 10, wherein said host cell is E. coli.
 12. A method according to claim 8, wherein said host cell is E. coli, and said peptide sequence homologous to said host cell is derived from E. coli β-galactosidase.
 13. A method of immunizing a host against infection with a parasitic nematode, comprising administering to said host a protein according to claim
 1. 14. A protein according to claim 5, wherein said protein further comprises the sequence:

    __________________________________________________________________________           Glu                                                                               Phe                                                                               Ala                                                                               Glu                                                                               Arg                                                                               Ser                                                                               Val                                                                               Gln                                                                               Lys                                                                               Leu                                                                               Gln                                                                               Lys                                                                               Glu                                                                               Val                               Asp                                                                               Arg                                                                               Leu                                                                               Glu                                                                               Asp                                                                               Glu                                                                               Leu                                                                               Val                                                                               His                                                                               Glu                                                                               Lys                                                                               Glu                                                                               Arg                                                                               Tyr                                                                               Lys                                                                               Ala                               Ile                                                                               Ser                                                                               Glu                                                                               Glu                                                                               Leu                                                                               Asp                                                                               Ser                                                                               Thr                                                                               Phe                                                                               Gln                                                                               Glu                                                                               Leu                                                                               Ser                                                                               Gly                                                                               Tyr.                                 __________________________________________________________________________


15. A vaccine comprising an effective amount of a protein according to claim 1 together with a pharmaceutically or veterinarally acceptable carrier or diluent.
 16. A vaccine according to claim 15 further comprising an adjuvant.
 17. A method of protecting a host against Trichostrongylus contortus infestation comprising administering to the host an effective amount of a protein according to claim
 1. 